CHIMERIC ANTIGEN RECEPTORS (CARs), COMPOSITIONS AND METHODS THEREOF

ABSTRACT

The present disclosure relates to compositions and methods relating to chimeric antigen receptor (CAR) polypeptides and methods relating thereto. In one embodiment, the present disclosure relates to engineered cells having chimeric antigen receptor polypeptides directed to at least two targets. In another embodiment, the present disclosure relates to engineered cells having chimeric antigen receptor polypeptides and an enhancer moiety.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/312,769, filed on Dec. 21, 2018, which claims priority based onInternational PCT Application No. PCT/US2016/068349, filed on Dec. 22,2016, and U.S. Provisional Application No. 62/369,004, filed on Jul. 29,2016, which is a continuation of International PCT Application No.PCT/US2016/039306, filed Jun. 24, 2016, which claims the benefit of U.S.Provisional Application No. 62/184,321, filed Jun. 25, 2015, U.S.Provisional Application No. 62/235,840, filed Oct. 1, 2015, and U.S.Provisional Application No. 62/244,435, filed Oct. 21, 2015, thecontents of which are incorporated herein by reference in its entirety.

BACKGROUND

T cells, a type of lymphocyte, play a central role in cell-mediatedimmunity. They are distinguished from other lymphocytes, such as B cellsand natural killer cells (NK cells), by the presence of a T-cellreceptor (TCR) on the cell surface. T helper cells, also called CD4+ Tor CD4 T cells, express CD4 glycoprotein on their surface. Helper Tcells are activated when exposed to peptide antigens presented by MHC(major histocompatibility complex) class II molecules. Once activated,these cells proliferate rapidly and secrete cytokines that regulateimmune response. Cytotoxic T cells, also known as CD8+ T cells or CD8 Tcells, express CD8 glycoprotein on the cell surface. The CD8+ T cellsare activated when exposed to peptide antigens presented by MHC class Imolecules. Memory T cells, a subset of T cells, persist long term andrespond to their cognate antigen, thus providing the immune system with“memory” against past infections and/or tumor cells.

T cells can be genetically engineered to produce special receptors ontheir surface called chimeric antigen receptors (CARs). CARs areproteins that allow the T cells to recognize a specific protein(antigen) on tumor cells. These engineered CAR T cells are then grown inthe laboratory until they number in the billions. The expandedpopulation of CAR T cells is then infused into the patient.

Clinical trials to date have shown chimeric antigen receptor (CAR) Tcells to have great promise in hematologic malignancies resistant tostandard chemotherapies. Most notably, CD19-specific CAR (CD19CAR)T-cell therapies have had remarkable results including long-termremissions in B-cell malignancies (Kochenderfer, Wilson et al. 2010,Kalos, Levine et al. 2011, Porter, Levine et al. 2011, Davila, Riviereet al. 2013, Grupp, Frey et al. 2013, Grupp, Kalos et al. 2013, Kalos,Nazimuddin et al. 2013, Kochenderfer, Dudley et al. 2013, Kochenderfer,Dudley et al. 2013, Lee, Shah et al. 2013, Park, Riviere et al. 2013,Maude, Frey et al. 2014).

Despite the success of CAR therapy in B-cell leukemia and lymphoma, theapplication of CAR therapy to T-cell malignancies has not yet been wellestablished. Given that T-cell malignancies are associated withdramatically poorer outcomes compared to those of B-cell malignancies(Abramson, Feldman et al. 2014), CAR therapy in this respect has thepotential to further address a great clinical need.

To date, current efforts have focused on CAR T-cells demonstratingefficacy in various B-cell malignancies. While initial remission ratesof approximately 90% are common in B-ALL using CD19CAR, most of theserelapse within a year. The relapse is at least in part due to theantigen escape. Thus, more effective CAR T cell treatments in order toprevent the relapse is urgently needed. Target discovery and selectionare the initial step as there are no general rules to ensure or guideCAR design that are efficacious.

There are some roadblocks that hinder the broader adoption of CARtherapeutic approach. Among the most general challenges are: (1)selection of antigen target and chimeric antigen receptor(s); (2) CARdesign; (3) tumor heterogeneity, particularly the variance in thesurface expression of tumor antigens. Targeting single antigen carriesthe risk of immune escape and this could be overcome by targetingmultiple desired antigens; (4) immunosuppressive microenvironment. CAR Tcells may be suppressed and de-activated on arrival at the tumor site.

Most CAR chimeric antigen receptors are scFvs derived from monoclonalantibodies and some of these monoclonal antibodies have been used in theclinical trials or treatment for diseases. However, they have limitedefficacy, which suggests that alternative and more potent targetingapproaches, such as CARs are required. scFvs are the most commonly usedchimeric antigen receptor for CARs. However, CAR affinity binding andlocations of the recognized epitope on the antigen could affect thefunction. Additionally the level of the surface CAR expression on the Tcells or NK cells is affected by an appropriate leader sequence andpromoter. Furthermore, overexpressed CAR proteins can be toxic to cells.

Therefore, there remains a need for improved chimeric antigenreceptor-based therapies that allow for more effective, safe, andefficient targeting of T-cell associated malignancies.

SUMMARY OF THE INVENTION

In one embodiment, the present disclosure provides an engineered cellhaving a first chimeric antigen receptor polypeptide including a firstantigen recognition domain, a first signal peptide, a first hingeregion, a first transmembrane domain, a first co-stimulatory domain, anda first signaling domain; and a second chimeric antigen receptorpolypeptide including a second antigen recognition domain, a secondsignal peptide, a second hinge region, a second transmembrane domain, asecond co-stimulatory domain, and a second signaling domain; wherein thefirst antigen recognition domain is different than the second antigenrecognition domain.

In another embodiment, the present disclosure provides an engineeredpolypeptide including a chimeric antigen receptor and an enhancer.

In another embodiment, the present disclosure provides an engineeredpolypeptide including a chimeric antigen receptor polypeptide and anenhancer.

In another embodiment, the present disclosure provides an engineeredchimeric antigen receptor polypeptide, the polypeptide including: asignal peptide, a CD45 antigen recognition domain, a hinge region, atransmembrane domain, at least one co-stimulatory domain, and asignaling domain. In another embodiment, the present disclosure providesa polynucleotide encoding for the aforementioned polypeptide.

In another embodiment, the present disclosure provides an engineeredcell having the engineered polypeptide or polynucleotide describedabove.

In another embodiment, the present disclosure provides a method ofreducing the number of target cells including the steps of (i.)contacting said target cells with an effective amount of an engineeredcell having at least one chimeric antigen receptor polypeptide, forengineered cells having multiple chimeric antigen receptor polypeptides,each chimeric antigen receptor polypeptide is independent; and (ii.)optionally, assaying for the reduction in the number of said cells. Thetarget cells include at least one cell surface antigen selected from thegroup consisting of interleukin 6 receptor, NY-ESO-1, alpha fetoprotein(AFP), glypican-3 (GPC3), BAFF-R, BAFF, APRIL, BCMA, TACI, LeY, CD5,CD13, CD14, CD15 CD19, CD20, CD22, CD33, CD41, CD45, CD61, CD64, CD68,CD117, CD123, CD138, CD267, CD269, CD38, Flt3 receptor, and CS1.

In another embodiment, the present disclosure provides methods fortreating B-cell lymphoma, T-cell lymphoma, multiple myeloma, chronicmyeloid leukemia, B-cell acute lymphoblastic leukemia (B-ALL), and cellproliferative diseases by administering any of the engineered cellsdescribed above to a patient in need thereof.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 . A schematic representation of cCAR construct (hereinafter,“multiple CAR or compound CAR”). Multiple or compound CAR targetsmultiple antigens (e.g. cell type 1 or cell type 2 or the same celltype). Multiple or cCAR T cell immunotherapies comprises individualcomponent CAR comprising a different or same antigen recognition domain,a hinge region, a transmembrane domain, various co-stimulatory domain(s)and an intracellular signaling domain.

FIGS. 2A-2C. A schematic representation of cCAR-T construct. (2A) Theconstruct comprises a SFFV promoter driving the expression of-multiplemodular units of CARs linked by a P2A peptide. Upon cleavage of thelinker, the cCARs split and engage upon targets expressing CD33 and/orCD123. As a novel cCAR construct, the activation domains of theconstruct may include, but is not limited to, 4-1BB on the CD33 CARsegment and a CD28 region on the CD123 CAR. (2B). A Western blotdepicting the expression of transduced CD33CD123 cCAR-T cells. Thefigure depicts expression of two different CAR proteins, i.e., CD33 CARand CD123 CARs. The cCAR-T cells expressing both CD33 and CD123 CARsupon cleavage of the linker generate two distinct and consistentlyintense protein bands. Green Fluroscent Protein (GFP) is included asnegative control. (2C). Flow cytometry representing the efficiency oftransduction. Upper panel shows the lentiviral titer for CD33CD123 cCARs(also referred to as CD33CD123-2G-CAR) tested on 293FT HEK (humanembryonic kidney) cells to gauge maximum transduction efficiency beforeusage on UCB (umbilical cord blood) and PB (peripheral blood) T-cells.Lower panel shows CD33CD123 cCAR (also referred to as CD33CD123-2G-CAR)T-cells transduced with lentiviral vectors comprising CD33CD123 cCARconstruct and GFP-transduced cells as control Percentages indicated byyellow circles are proxies for transduction efficiency.

FIG. 3 . Schematic showing a method of generating a high-efficiencycompound CAR (cCAR). HEK-293-FT cells are transfected with compound CARplasmid DNA and lipofectamine 2000; viral supernatant collected at about36 hr and at about 60 h; filtered and stored at −80° C. T cells areactivated with anti-mouse CD3 antibody and IL-2 for at least 2 days.Activated T cells are transduced at least once with thawed lentivirus onretronectin-coated plates; After at least one overnight transductions at0.3×106 T cells/mL for about 2 days, the number of T cells was reducedin order to increase transduction efficiency. After transduction, cellsare washed and expanded; Flow analysis (F(Ab′)2 labeling) is done toconfirm CAR efficiency on day 3; total 5-7 day expansion. cCAR T cellsare co-cultured with target cells in vitro and cCAR T cells killingefficacy of cancer cells is assessed in vivo (mice).

FIG. 4 . A co-culture assay representing the incubation of CD33CD123-2GCAR-T cells (cCAR) with the promyelocytic leukemia cell line HL60.cCAR-T cell (lower panel) is compared to control GFP transduced T-cell(upper panel). The efficacy of the killing is measured by the populationof CD33+ cells that is left over after incubation for about 24 hours(enclosed in yellow circles).

FIG. 5 . A co-culture assay representing incubation of cCAR-T cells withthe myelogenous leukemia cell line KG-1a, which expresses about 100%CD33 and about 50-80% CD123. cCAR-T cell (lower panel) is compared tocontrol GFP transduced T-cell (upper panel). The efficacy of the killingis measured by the population of CD33+ cells that is left over afterincubation for about 24 hours.

FIG. 6 . CD33CD123 cCAR-T cells co-cultures with AML-9 at 5:1. Aco-culture assay representing incubation of cCAR-T cells with AMLpatient samples (here referred to as AML-9). The patient cells includemixed populations of cells, such as for example, leukemia cells,monocytes, and other types of blasts. CD33 acts as a marker for CAR-Taction as well as CD34, a specific marker for leukemia cells. The CAR-Tpanel (right) is compared to control GFP transduced T-cells (middle).The efficacy of the killing is measured by the population of CD33+/CD34+cells that is left over after incubation for at least 24 hours.

FIG. 7 . CD33CD123 cCAR-T cells co-cultures with Sp-BM-B6 at 5:1. Aco-culture assay representing incubation of cCAR-T cells with B-ALLpatient samples (here referred to as Sp-BM-B6). The patient cellsinclude mixed populations of cells, such as, for example, leukemiacells, monocytes, and other types of blasts. CD34 acts as a specificmarker for leukemia cells. The CAR-T panel (right) is compared tocontrol GFP transduced T-cells (middle). The efficacy of the killing ismeasured by the population of CD34+ cells left over after incubation forat least 24 hours.

FIG. 8 . CD33CD123 cCAR expression in NK-92 cells. The CD33CD123 cCARexpression are detected using goat-anti-mouse antibody, F(ab)2.

FIG. 9 . A co-culture assay representing incubation of CD33CD123 cCARNK-92 cells with HL-60. The cCAR NK-92 cells are compared with GFPtransduced NK-92 cells. The efficacy of the killing is measured by thepopulation of CD33+ cells left over after incubation for about 24 hours.

FIG. 10 . A co-culture assay representing incubation of cCAR NK-92 cellswith KG1a. The cCAR NK cell panel is compared with GFP transduced NK-92cells. The efficacy of the killing is measured by the population ofCD33+ cells left over after incubation for about 24 hours.

FIG. 11 . Dose response of CD33CD123 cCAR (CAR-CD33/123) NK-92 cellswith HL-60 or KG1a. The efficacy of the killing is measured by thepopulation of CD33+ cells left over after incubation for about 24 hours.

FIG. 12 . A comparison of CD33CD123 cCAR NK-92 cell killing ability withcontrol in two populations of KG11 cells. Assays were performed atdifferent ratios of CAR-CD33/123 (CD33CD123 cCAR NK-92 cells) and targetcells, kG1a. The efficacy of the killing is measured by the populationof CD33+CD123+ or CD33+CD123− cells left over after incubation for about24 hours.

FIGS. 13A-13B. Links by P2A and T2A schematic showing both cCAR-T and4-1BBL in a single construct. (13A) The construct consists of a SFFVpromoter driving the expression of two modular units of CARs A peptideand an enhancer, 4-1BBL. Upon cleavage of the linkers, the cCARs and4-1BBL split and engage upon targets expressing CD33 and/or CD123 and4-1BBL. Compound CAR, CD33CD123 CAR T cells received not onlycostimulation through the CD28 but also 4-1BB ligand (4-1BBL or CD137L).The CD3-zeta signaling domain complete the assembly of this CAR-T.(13B). Expression the CD33CD123-41BBL-2G construct in T-cells. T-cellsderived from peripheral blood from healthy donors were transduced withthe CD33CD123-4-1BBL-2G construct in 6-well plates incubated with 2 mlof virus supernatant. CAR expression was assayed with F(ab)′ labelingfor surface expression of the CAR protein and subsequently underwentFACS analysis. Transduced cells were compared to control T-cells labeledat the same time. Expression was determined and transduced populationencircled on plot 1 day after end of transduction period.

FIG. 14 . Links by P2A and T2A schematic showing both cCAR-T andIL-15/IL-15sushi in a single construct. The construct consists of a SFFVpromoter driving the expression of two modular units of CARs and anenhancer, IL-15/IL-15sushi. Upon cleavage of the linkers, the cCARs andIL-15/IL-15sushi split and engage upon targets expressing CD33 and/orCD123. The CD3-zeta signaling domain completes the assembly of thisCAR-T. The enhancers include, but not limited to, IL-15/IL-15sushi oncCAR.

FIG. 15 . A schematic representation of cCAR. The construct comprises aSFFV promoter driving the expression of multiple modular units of CARslinked by a linker. Upon cleavage of the linker, the cCARs split andengage upon targets expressing combinations of various target antigens:CD19 and/or CD20, and/or CD22 and/or 138. Multiple cCARs utilize thesame or different co-stimulatory domains, such as, without limiting4-1BB (also labeled as 4-BB) and/or CD28.

FIGS. 16A-16C. Activated T cells transduced to make CD19CD20-2G,CD19CD22-2G CAR T cells (all are L8). (16A) Design of compound CARs.(16B) Western blot. HEK-293T cells were transfected with lentiviralplasmids for control vector (lane 1), CD19CD20-2G (lane 2), andCD19CD22-2G (lane 3). 48 hours after transfection, supernatant wasremoved, and cells were also harvested. Cells were lysed for Westernblot and probed with mouse anti-human CD3z primary antibody, and goatanti-mouse HRP secondary antibody. (16C) PMBC buffy coat T cells wereactivated 3 days with anti-CD3 antibody. Cells were transduced witheither control vector (left), CD19CD20-2G (middle), or CD19CD22-2G,(right) lentiviral supernatant. After 3 days of incubation, cells wereharvested and incubated with goat anti-mouse Fab2 or goat IgG antibodiesconjugated with biotin for 30 minutes. Cells were washed, suspended andstained with streptavidin-PE and mouse anti-human CD3-PerCp for 30minutes. Cells were washed and suspended in 2% formalin, and analyzed byflow cytometry to determine CAR efficiency. (N=2).

FIG. 17 . Expression of compound CD19CD22CAR T cells using differentleader sequences. PMBC buffy coat T cells were activated 3 days withanti-CD3 antibody. Cells were transduced with either control vector(left), L8-CD19CD22-2GCAR (middle left), L45-CD19CD22-2GCAR, (middleright) or CSF-CD19CD22-2GCAR (right) lentiviral supernatant. Thesupernatants were each 3× concentrated. After 3 days of incubation,cells were harvested and incubated with goat anti-mouse Fab2 or goat IgGantibodies conjugated with biotin for 30 minutes. Cells were washed,suspended and stained with streptavidin-PE and mouse anti-humanCD3-PerCp for 30 minutes. Cells were washed and suspended in 2%formalin, and analyzed by flow cytometry to determine CAR efficiency.(N=2).

FIG. 18 . Comparison of transduction efficiency using concentrated vsunconcentrated L8-CD19CD22-2G or L8-CD19CD20-2G lentiviral supernatant.A. PMBC buffy coat T cells were activated 3 days with anti-CD3 antibody.Cells were transduced with either control vector (left), unconcentrated(middle) L8-CD19CD22-2GCAR or 3× concentrated L8-CD19CD22-2GCAR (right)lentiviral supernatant. After 3 days of incubation, cells were harvestedand incubated with goat anti-mouse Fab2 or goat IgG antibodiesconjugated with biotin for 30 minutes. Cells were washed, suspended andstained with streptavidin-PE and mouse anti-human CD3-PerCp for 30minutes. Cells were washed and suspended in 2% formalin, and analyzed byflow cytometry to determine CAR efficiency. (N=2). B. The sameexperiment was useed for constructs containing L8-CD19CD20-2Gunconcentrated or 2.5× concentrated lentiviral vector.

FIG. 19 . L8-CD19CD22-2G CAR T cells lyse SP53 tumor cells in overnightco-culture. Activated PMBC T cells transduced with either control (toprow), L8-CD19CD22-2G, or (bottom row) lentiviral supernatant wereincubated with SP53 cells at the ratios of 1:1 (left) 2:1 (middle) and5:1 (right), effector:target cells. After 24 hours of incubation at 37°C., samples were washed and stained with anti-human CD3-PerCp andanti-human CD19-APC, washed, and analyzed by flow cytometry. SP53 cellsalone are shown on the far upper right, and a summary of percent lysisat each ratio is on the lower right. (N=2).

FIG. 20 . L8-CD19CD22-2G CAR T cells lyse JeKo-1 tumor cells inovernight co-culture. Activated PMBC T cells transduced with eithercontrol (left), or L8-CD19CD22-2G, (middle) 3× concentrated lentiviralsupernatant were incubated with JeKo-1 cells at the ratios of 2:1 (top)and 5:1 (bottom), effector:target cells. After 24 hours of incubation at37° C., samples were washed and stained with anti-human CD3-PerCp andanti-human CD19-APC, washed, and analyzed by flow cytometry. JeKo-1cells alone and a summary of cell lysis are shown on the right. (N=2).

FIG. 21 . L8-CD19CD22-2G CAR T cells lyse AML patient cells in overnightco-culture. Activated PMBC T cells transduced with either control(left), or L8-CD19CD22-2G, (middle) 3× concentrated lentiviralsupernatant were incubated with CMTMR-stained cells from a patientdiagnosed with AML (PT1) at the ratios of 2:1 (top) and 5:1 (bottom),effector:target cells. After 24 hours of incubation at 37° C., sampleswere washed and stained with anti-human CD3-PerCp and anti-humanCD19-APC, washed, and analyzed by flow cytometry. Patient cells aloneand a summary of cell lysis are shown on the right. (N=2).

FIGS. 22A-22B. L8-CD19CD22-2G CAR T cells deplete CD19+B-ALL patientcells. (22A) Activated PMBC T cells transduced with either control(left), or L8-CD19CD22-2G, (middle) lentiviral supernatant wereincubated with CMTMR-stained cells from a patient with B-ALL (PT2) at a1:1 ratio for 4 days in the presence of 2.5% FBS and IL-2. Followingthis incubation at 37° C., samples were washed and stained withanti-human CD3-PerCp and anti-human CD19-APC, washed, and analyzed byflow cytometry. Prestained patient cells cultured alone for 4 days areshown on the right. (22B). L-8-CD19CD22-2G cCAR T-cells show effect onCD22⁺ K562 cells. An artificial K562 expressing CD22 cell line(K562xp22) via transduction into wild-type K562 cells was generated.Subsequently, we tested the anti-tumor properties of the CD19CD22 cCARto target the minor CD22⁺ population of the K562 cells. A co-cultureexperiment at 1:1 ratio (effective:target) show a modest significantcytotoxic effect on K562 expressing CD22 population compared to thecontrol. Co-cultures were stained with CD3, CD19 and CD22 to separateeffector and target populations by flow cytometry. The result wasgraphed. Cytotoxicity results remain consistent with other numbersreported for anti-tumor activity against artificial antigen presentingcell lines.

FIGS. 23A-C. Various transduction schemes for BC1cCAR lentivirus. (23A)Method 1 consisting of a 2×transduction for 24 hours each time is abaseline transduction scheme. Scheme proceeds according to the figure.(23B) Method 2 possesses the same methodology as Method 1, however, thesecond transduction is replaced by continued incubation. (23C) Method 2revised uses viral supernatant incubated with cells directly for 48hours.

FIGS. 24A-24D: CAR construct scheme and comparison of transductionmethodologies. (24A) BC1cCAR's modular design consists of an anti-CD269(BCMA) single-chain variable fragment (scFv) region fused to ananti-CD319 (CS1) scFv by a self-cleaving P2A peptide, CD8-derived hinge(H) and transmembrane (TM) regions, and tandem CD28 and 4-1BBco-activation domains linked to the CD3ζ signaling domain. A strongspleen focus forming virus promoter (SFFV) and a CD8 leader sequencewere used for efficient expression of the CD3CAR molecule on the T-cellsurface. (24B) BC1cCAR's expression was measured via flow cytometryagainst an isotype control. Population encircled represents transducedCAR cells. (24C) Transduction efficiency is improved by optimal methods.(24D). Protein expression of BC1cCAR and BCMA-CS1-2G in HEK-293FT cells.HEK-293FT cells were transfected with lentiviral plasmids for GFP (lane1), BC1cCAR (lane 2) 48 hours after transfection, supernatant wasremoved, and cells were also removed. Cells were lysed for Western blotand probe with mouse anti-human CD3z antibody. C. Transductionefficiency is improved by optimal methods.

FIGS. 25A-25C. in vitro evaluation of BC1cCAR T-cells against myelomacell lines. (25A) BC1cCAR and control T-cells were cultured with highlyBCMA positive MM1S and RPMI-8226 cells for 24 hours at E:T ratios of 2:1and 5:1. Target MM1S and RPMI-8226 cells were stained by Cytotracker dye(CMTMR) to distinguish it from effector T-cells. Populations were gatedby anti-BCMA (CD269) and anti-CS1 (CD319 antibodies) along withCMTMR-PE. Target U266 cells were labeled with Cytotracker (CMTMR) dye todistinguish it from effector T-cells. Encircled populations representtumor cells. (25B) U266 target depletion. BC1cCAR and control T-cellswere also incubated with U266 cells expressing BCMA and a subset of CS1.Target tumor cells were stained as described above and gating conditionsapplied similarly. Tumor populations are encircled. (25C) In vitrosummary of BC1cCAR T activity against human myeloma cell lines.Graphical summary of BC1cCAR T-cell in vitro cytotoxicity againstvarious myeloma cell lines at 2:1 and 5:1 E:T ratios.

FIGS. 26A-26D. Characterization of BC1cCAR T-cell anti-tumor activityagainst primary myeloma tumor cells. (26A) Dose dependent effect onMM7-G primary double phenotype tumors. BC1cCAR and control T-cells werecultured against BCMA⁺CS1⁺ primary myeloma cells MM7-G for 24 hours.Target cells were pre-stained with CMTMR and cultures were carried outin E:T ratios of 2:1, 5:1, and 10:1. Populations were gated by BCMA andCS1, along with CMTMR, and flow cytometry plots with populationsencircled represent target tumor populations (left). Bar graphsummarizing in vitro cytotoxicity is shown for clarity (right). (26B)Population specific depletion in MM10-G. Co-cultures with MM10-G primarytumor cells were carried out in similar conditions. When stained withanti-CS1 and anti-BCMA antibody, MM10-G reveal distinct populations.BCMA⁺CS1⁺ double positive populations are colored purple whilst CS1⁺only populations are colored dark blue. BC1cCAR T-cell cytotoxicityagainst each population is summarized in the bar graph below. (26C) Dosedependent effect on CS1dim BCMAneg. MM11-G primary tumor. A thirdexperiment using BCMA^(dim)CS1^(dim) primary cells (MM11-G) furthershows BC1cCAR cytotoxicity effects over a range of E:T dosagessummarized. (26D) Summary panel graph showing BC1cCAR T-cellcytotoxicity against myeloma cell lines and primary tumor cells with avariety of BCMA and CS1 compositions.

FIGS. 27A-27D. Functional validation of BC1cCAR antigenic specificity.(27A) We engineered a CML cell line, K562, to express either BCMA or CS1independently. Wild-type K562 shows as a negative peak. while BCMAexpressing K562 (BCMAxpK562) and CS1 expressing K562 (CS1xpK562) showpopulation shifts in their respective antigen expression ranges. (27B)Short term (4 hour-12 hour) cultures of BC1cCAR T-cells against eitherBCMAxpK562 or CS1xpK562 show antigen specific cytotoxicity correlatingwith E:T dosage increase. Experiments against wild-type K562 wereperformed as a negative control A CS1-specific single CAR was generatedto compare efficacy with BC1cCAR against CS1xpK562 cells and aredelineated with red bars in the respective plot. Anti-CS1 specificactivity was also seen against CS1^(dim) NK-92 cells after 24 hours ofculture. (27C) Comparison between single antigen CARs and BC1cCAR T inmixed cell assays. Long-term cultures were conducted over a 48 hourperiod with a 5:1 mixture of BCMAxpK562 cells and CS1xpK562 cells.BC1cCAR, CS1-CAR, BCMA-CAR, and control T-cells were added at a 5:1 E:Tratio to each treatment well and flow cytometry analyses acquired.Histogram plots showing residual populations of BCMA or CS1 cells areshown per treatment condition, with red lines demarcating T-cell ortarget tumor populations. Numerical values in histogram plots representresidual gated populations of target tumor cells. (27D) BC1cCAR Tactivity against CS1 subsets in primary bone-marrow aspirate. Furtherco-culture experiments were conducted using bone-marrow aspirate samplesas CS1 expressing minority subsets. BC1cCAR or control T-cells wereadded at 2:1 (left panel), 5:1 (middle panel), or 10:1 (right panel)ratios and encircled populations represent target CS1 expressingpopulations. Results are analyzed by flow cytometry (upper). Summarygraph of anti-CS1 activity against bone marrow subsets (below).

FIGS. 28A-28C: Long-term sequential killing assay and tumorre-challenge. (28A) Scheme for construction of long-term sequentialkilling assay. Assay was conducted over a period of 168 hours with noexogenous cytokines where the initial culture was set-up with a 1:1 E:Tratio of CAR cells or control cells mixed with MM1S tumor cells. After48 hours, flow cytometry analysis was acquired for a small samplecollection and MM1S cells re-introduced into each treatment well.Repeated until the 168 hour time-point. (28B) T-cell proliferation andresponse after 48 hours. Images were taken on day of flow cytometryacquisition and cells were stained with anti-BCMA, anti-CS1, andanti-CD3 antibodies MM1S cells express as highly BCMA⁺ with a large CS1⁺proportion. Encircled populations represent the MM1S tumor presence,colored blue. (28C) CAR cell proliferation and antigen depletion after108h. Similar image acquisition and flow cytometry analysis wasperformed at the 108 hour time mark.

FIGS. 29A-29E. BC1cCAR T-cells demonstrate anti-leukemic effects invivo. (29A) IVIS imaging of MM1S Luc+ injected mouse model. NSG micewere sublethally irradiated and intravenously injected withluciferase-expressing MM1S multiple myeloma cells to induce measurabletumor formation. After 3 days, the mice were intravenously injected with5×10⁶ BC1cCAR T-cells or control GFP T-cells. On days 3, 6, 8 and 11,mice were injected subcutaneously with RediJect D-Luciferin andsubjected to IVIS imaging. (29B) BC1cCAR T-cells control MM1S tumorgrowth. Average light intensity measured for the BC1cCAR T-cellsinjected mice was compared to that of GFP control T-cell injected mice.(29C) BC1cCAR T-cells improve murine survival outlook. Percent survivalof mice was measured and compared between the two groups and log-rankmantel-cox test was conducted to calculate significance of improvedsurvival outlook. (29D). BCMA-CAR and BC1 cCAR T-cells demonstrate aprofound anti-leukemic effect on a mixture of K562 cells expressing BCMAand CS1 in xenograft mouse model. Luciferase positive K562 cellsexpressing BCMA are mixed with luciferase positive K562 cells expressingCS1 at a ratio of 4:1 BCMA to CS1 K562 cells. The mixed K562 cells(0.5×10⁶ cells) were then injected intravenously (day 1) at 24 h laterafter sub-lethal irradiation. After day 3, a course of BCMA CAR T-cells,BC1cCAR T-cells or control T-cells were intravenously injected into eachmouse (n=5 for each group). Dorsal side of tumor burden was measuredusing IVIS imaging system at days 3, 7, 10 and 12. At day 7 BCMA mouse#3 has large tumor. At day10 Dorsal BCMA vs control=47.7% less tumor,cCAR vs control=53.8% less tumor. At day 12 RESULTS (ventral view only)Dorsal BCMA vs control=43.8% less tumor, cCAR vs control=60.7% lesstumor. (29E). BCMA and BC1 cCAR T-cells in vivo significant reduction oftumor burden. Percent reduction relative to control in mice treated withBCMA CAR T-cells or cCAR (BC1 cCAR) relative to control over time.

FIGS. 30A-30B: BC1cCAR transduction into NK-92 cells. (30A) BC1cCAR'smodular design is comprised as shown and described previously. (30B) CARexpression on NK-92 cell surface. The construct was transduced intoNK-92 cells by incubating with viral supernatant for 48 hours andlabeling with F(ab)′ antibody detection for CAR protein surfaceexpression. Transduced populations are encircled and compared to controlNK-92 cells.

FIGS. 31A-31C. Characterization of BC1cCAR NK-92 anti-tumor properties.(31A) BC1cCAR NK cells lyse myeloma cell lines and primary cells.BC1cCAR NK-92 cells were incubated against U266, RPMI-8226, and MM1Smyeloma cell lines in addition to primary MM7-G tumor cells. Co-cultureswere carried out over 2 hours at an E:T ratio of 5:1 and labeled withanti-CS1 and anti-BCMA antibodies to separate out populations. Tumorpopulations are encircled. MM7-G primary tumor cells were stained withcell cytotracker dye (CMTMR) to distinguish from NK-92 cells and areencircled. Summary bar graph of BC1cCAR NK-92 cytotoxic activity ispresented as a visualization aid. (31B) BC1cCAR NK-92 cells were testedfor antigen specific activity using artificially generated BCMAexpressing K562 (BCMAxpK562) and CS1 expressing K562 (CS1xpK562) cells.(31C) Co-cultures were carried out over 4 hours at an E:T ratio of 5:1.K562 populations were previously stained with CMTMR and encircled in theflow cytometry plots. Bar graph summarizing anti-tumor activity tovisualize.

FIGS. 32A-32C. Generation and characterization of different BAFF-CARconstructs. (32A) L45-BAFF-28 CAR expression T-cell surface. L45-BAFF-28CAR was transduced into T-cells and evaluated for surface expressionusing F(ab)′ antibody. Gating was compared to controls. (32B) CARexpression dependence on leader sequence. BAFF-CAR constructs usingdifferent leader sequences were tested to determine if efficiency intransduction could be improved. Surface detection was evaluated usingF(ab)′ antibody and transduced populations encircled. (32C) CARexpression dependence on construct design. Additional BAFF-CARconstructs containing different leader sequences and construct designs(additional units) were validated and used to determine if CARtransduction could be improved. Transduced populations are encircled andgating compared to control T-cells. CSF-BAFF-28 41BBL is a BAFF CARco-expressing 4-1BBL (41BBL) with a CSF leader sequence. CSF-BAFF-28IL-15RA is a BAFF CAR co-expressing IL-15/IL-15sushi (IL-15RA) with aCSF leader.

FIG. 33 : Characterization of L45-BAFF-28 CAR T anti-tumor properties.L45-BAFF-28 CAR T-cells possess anti-tumor activity against MM1S tumorcells. L45-BAFF-28 CAR T-cells were cultured for 48 hours at an E:Tratio of 3:1 against MM1S myeloma cells. Duplicate samples are shown.Cytotoxic activity is summarized in the bar graph.

FIGS. 34A-34C: Characterization of anti-tumor activity using differentBAFF-CAR constructs and enhancements. (34A) BAFF-CAR constructs againstMM1S cells. L8-BAFF-28IL-15/IL-15sushi and L8-BAFF-28-41BBL CARs werecultured for 24 hours against MM1S tumor cells at an E:T ratio of 5:1.Tumor populations are encircled. (34B) BAFF-CAR constructs against SP53cells. Both CARs and L45-BAFF-28 CAR were cultured against Sp53 tumorcells (B-lineage) at an E:T ratio of 5:1 for 24 hours. (34C) Summary bargraph of cytotoxic activity.

FIG. 35 . A schematic showing cCAR construct. The construct consists aSFFV promoter driving the expression of two modular units of CAR linkedby a P2A peptide. Upon cleavage of this P2A peptide, the cCARs split andengage upon targets expressing BCMA and/or CD19. Two unit CARs usedifferent or same co-stimulatory domain. A co-stimulatory domain couldbe, but limited to, 4-1BB or CD28.

FIGS. 36A-36B. Characterization of the BCMA CAR unit. (36A) BCMA CAReffectively deplete BCMA+ MM1S cells. The BCMA CAR was transduced intoT-cells and co-cultured with MM1S tumor cells. A CS1 CAR was alsogenerated and used for robustness. MM1S cells are significantly dualpositive for both BCMA and CS1. Co-cultures were conducted over 48 hourswith BCMA and CS1 antibodies used to identify tumor centers. Encircledpopulations represent residual MM1S tumor cells after culture. (36B)BCMA CAR effectively lyses BCMA+ primary tumor cells. (36B) The BCMA CARand CS1 CAR were also evaluated for its anti-tumor properties againstprimary MM7-G myeloma patient cells. The MM7-G population is a majorityBCMA⁺ CS1⁺ population with minority but significant CS1⁺ onlypopulations as well. Both BCMA CAR and CS1 CAR were used in tandem toevaluate cytotoxicity with BCMA and cytotracker (CMTMR) used todifferentiate tumor populations from CAR cells.

FIGS. 37A-37C. Characterization of CD19 CARs. (37A) Design of CD19CARunit. (37B) Western blot. HEK-293T cells were transfected withlentiviral plasmids for control vector (lane 1) and CD19-2G (lane 2). 48hours after transfection, supernatant was removed, and cells were alsoharvested. Cells were lysed for Western blot and probed with mouseanti-human CD3z primary antibody, and goat anti-mouse HRP secondaryantibody. (37C). PMBC buffy coat T cells were activated 3 days withanti-CD3 antibody. Cells were transduced with either control vector(left), L8-CD19-2G (middle left), L8-CD19CD20-2G, (middle right) orL8-CD19CD22-2GCAR (right) lentiviral supernatant. After 3 days ofincubation, cells were harvested and incubated with goat anti-mouse Fab2or goat IgG antibodies conjugated with biotin for 30 minutes. Cells werewashed, suspended and stained with streptavidin-PE and mouse anti-humanCD3-PerCp for 30 minutes. Cells were washed and suspended in 2%formalin, and analyzed by flow cytometry to determine CAR efficiency.(N=2)

FIGS. 38A-38B. Expression of compound CD19CAR T cells using differentleader sequences. (38A) CAR constructs were designed to express thefusion protein with different leader sequences. (38B) PMBC buffy coat Tcells were activated 3 days with anti-CD3 antibody. Cells weretransduced with either control vector (left), HA-CD19-2G (top middle).IL2-CD19-2G (top right), L8-CD19-2G (lower middle left), L45-CD19-2G,(lower middle right) or CSF-CD19-2GCAR (lower right) lentiviralsupernatant. After 3 days of incubation, cells were harvested andincubated with goat anti-mouse Fab2 or goat IgG antibodies conjugatedwith biotin for 30 minutes. Cells were washed, suspended and stainedwith streptavidin-PE and mouse anti-human CD3-PerCp for 30 minutes.Cells were washed and suspended in 2% formalin, and analyzed by flowcytometry to determine CAR efficiency. (N=2) FIGS. 39A-39B. Expressionof CD19CAR on T cells using different CD19 scFv sequences. (39A) CARconstructs were designed to express the fusion protein with differentscFv sequences. (39B) PMBC buffy coat T cells were activated 3 days withanti-CD3 antibody. Cells were transduced with either control vector(left), L8-CD19-2G (middle), IL2-CD19-2G (top right), or L8-CD19b-BB-2G(right) lentiviral supernatant. After 3 days of incubation, cells wereharvested and incubated with goat anti-mouse Fab2 or goat IgG antibodiesconjugated with biotin for 30 minutes. Cells were washed, suspended andstained with streptavidin-PE and mouse anti-human CD3-PerCp for 30minutes. Cells were washed and suspended in 2% formalin, and analyzed byflow cytometry to determine CAR efficiency. (N=2) FIG. 40 . L8-CD19-2Gand CD19b-BB CAR T cells lyse SP53 tumor cells in overnight co-culture.Activated PMBC T cells transduced with either control (left),L8-CD19-2G, (middle) or L8-CD19b-BB-2G (right) lentiviral supernatantwere incubated with SP53 cells at the ratios of 2:1 (top) and 5:1(bottom), effector:target cells. After 24 hours of incubation at 37° C.,samples were washed and stained with anti-human CD3-PerCp and anti-humanCD19-APC, washed, and analyzed by flow cytometry. SP53 cells alone and asummary of cell lysis are shown on the far right. (N=2) FIG. 41 .L8-CD19-2G and CD19b-BB CAR T cells lyse JeKo-1 tumor cells in overnightco-culture. Activated PMBC T cells transduced with either control(left), L8-CD19-2G, (middle) or L8-CD19b-BB-2G (right) lentiviralsupernatant were incubated with JeKo-1 cells at the ratios of 2:1 (top)and 5:1 (bottom), effector:target cells. After 24 hours of incubation at37° C., samples were washed and stained with anti-human CD3-PerCp andanti-human CD19-APC, washed, and analyzed by flow cytometry. JeKo-1cells alone and a summary of cell lysis are shown on the far right.(N=2).

FIG. 42 . L8-CD19-2G and L8-CD19b-BB-2G CAR T cells lyse AML patientcells in overnight co-culture. Activated PMBC T cells transduced witheither control (left), L8-CD19-2G, (middle) or L8-CD19b-BB-2G (right)lentiviral supernatant were incubated with CMTMR-stained cells from apatient with AML at the ratios of 2:1 (top) and 5:1 (bottom),effector:target cells. After 24 hours of incubation at 37° C., sampleswere washed and stained with anti-human CD3-PerCp and anti-humanCD19-APC, washed, and analyzed by flow cytometry. Prestained patientcells alone and a summary of cell lysis are shown on the far right.(N=2).

FIG. 43 . L8-CD19-2G and L8-CD19b-BB-2G CAR T cells deplete CD19+patient cells. Activated PMBC T cells transduced with either control(left), L8-CD19-2G, (middle) or L8-CD19b-BB-2G (right) lentiviralsupernatant were incubated with CMTMR-stained cells from a patient withB-ALL. L8-CD19-2G T cells were incubated with patient cells at a 1:1ratio for overnight (top), while L8-CD19b-BB-2G T cells were incubatedwith patient cells at a 5:1 ratio for 40 hours (bottom). Following thisincubation at 37° C., samples were washed and stained with 5 anti-humanCD3-PerCp and anti-human CD19-APC, washed, and analyzed by flowcytometry. Prestained patient cells alone are shown on the far right.(N=2).

FIG. 44 . A schematic showing cCAR construct. The construct consists aSFFV promoter driving the expression of two modular units of CAR linkedby a P2A peptide. Upon cleavage of this P2A peptide, the cCARs split andengage upon targets expressing BCMA and/or CD19b. Two unit CARs usedifferent or same co-stimulatory domain. A co-stimulatory domain couldbe 4-1BB or CD28.

FIGS. 45A-45C. Generation and characterization of different BAFF-CARconstructs. (45A) L45-BAFF-28 CAR was transduced into T-cells andevaluated for surface expression using F(ab)′ antibody. Gating wascompared to controls. (45B) BAFF-CAR constructs using different leadersequences were tested to determine if efficiency in transduction couldbe improved. Surface detection was evaluated using F(ab)′ antibody andtransduced populations encircled. (45C) Additional BAFF-CAR constructscontaining different leader sequences and construct designs (additionalunits) were validated and used to determine if CAR transduction could beimproved. Transduced populations are encircled and gating compared tocontrol T-cells. CSF-BAFF-28 41BBL is a BAFF CAR co-expressing 4-1BBL(41BBL) with a CSF leader sequence. CSF-BAFF-28IL-15/IL-15sushi—is aBAFF CAR co-expressing IL-15/IL-15sushi with a CSF leader.

FIGS. 46A-46B: L45-BAFF-28 CAR T-cells possess anti-tumor activityagainst MM1S tumor cells. Characterization of L45-BAFF-28 CAR Tanti-tumor properties. (46A) BAFF CAR cytotoxic activity in vitrosummarized from (46B). (46B) L45-BAFF-28 CAR T-cells possess anti-tumoractivity against MM1S tumor cells. L45-BAFF-28 CAR T-cells were culturedfor 48 hours at an E:T ratio of 3:1 against MM1S myeloma cells.Duplicate samples are shown.

FIGS. 47A-47B Characterization of anti-tumor activity using differentBAFF-CAR constructs and enhancements. (47A) L8-BAFF-28IL-15/IL-15sushiand L8-BAFF-28 4-1BBL CARs were cultured for 24 hours against MM1S tumorcells at an E:T ratio of 5:1. Tumor populations are encircled. (47B)Both CARs and L45-BAFF-28 CAR were cultured against Sp53 tumor cells(B-lineage) at an E:T ratio of 5:1 for 24 hours.

FIG. 48 . CRISPR/Cas9 interference system. The expression of sgRNA andCas9 puromycin is driven by the U6 and SFFV promoters, respectively. TheCas9 is linked with puromycin resistant gene by E2A self-cleavingsequences.

FIGS. 49A-49B. (49A) Steps of generation of CAR T or NK cell targetinghematologic malignancies. (49B). Generation and cell sorting of stableCD45 knockdown NK-92 cells using CRISPR/Cas9 lentivirus system. Flowcytometry analysis indicated the CD45 expression levels on NK-92 cellsurface (left panels). After transduced sgCD45B CRISPR into NK-92 cells,transduced cells were cultured in medium containing puromycin for a fewweeks. CD45 negative NK-92 cells were determined using CD45 antibody andwere sorted. The purity of stable NK^(45i)-92 (CD45 knockdown) NK-92cells were determined by Flow cytometry analysis (right panel). Thisdata showed that we successfully generated and obtained NK^(45i)-92cells.

FIG. 50 . Cell growth curve of wild type, GFP transduced NK-92 orNK^(45i)-92NK cells. To evaluate the effect for cell proliferationcaused by CD45-knockdown (KD) in NK-92 cells, the number of cells ofNK-92(●), GFP-transduced NK-92(▪) and NK^(45i)-92(▴) were counted at 48h and 96 h after seeding into 24 well plates. IL-2 was added at 48 htime point. (n=3 independent experiments performed in duplicate). Dataare mean±S.D. These data indicated that knockdown of CD45 receptor onNK-92 show similar cell growth curve compared to non-transduced NK-92 orGFP-transduced NK-92 cells. 24 well, duplicate, n=3 IL-2 was added at 48hr time point.

FIGS. 51A-51B. Co-culture assay with CCRF-CEM (target: T) and GFP NK-92or GFP NK^(45i)-92 cells (effector: E) at 5:1 (E:T) ratio and 16 hourincubation. (51A) Flow cytometry analysis of CCRF-CEM only (blue dot inleft panel), in co-culture with CCRF-CEM and control GFP transducedNK-92 cells (middle panel) or GFP NK^(45i)-92 cells (right panel). Bluedots in all of panels indicates the leftover target CCRF-CEM cells andred dots shows effector cells by co-culture assay. The majority of theblue dots are in the upper left square of each experiment. All ofincubation time were 16 h and the ratio of effector T-cells: target cellis 5:1. All experiments were performed in duplicate. (51B) Bar graphindicates the percent of cell lysis by the GFP transduced NK^(45i)-92cells compared to the control GFP transduced NK92 cells in co-cultureassay with CCRF-CEM. These data suggest that knockdown of CD45 in NK-92cells does not show a significant difference for killing activityagainst CCRF-CEM cells compared to GFP-control NK-92 cells in vitroco-culture assay.

FIGS. 52A-52B. Co-culture assay with CCRF-CEM (target: T) and GFP NK-92,CD5CAR NK-92 or CD5CAR NK^(45i)-92 cells (effector: E) at 5:1 (E:T)ratio and 16 hour incubation. (52A) Flow cytometry analysis of CCRF-CEMonly (left panel), in co-culture with CCRF-CEM and control GFP NK-92cells (middle left panel), CD5CAR NK-92 cells (middle right panel),CD5CAR NK^(45i)-92 cells (right panel) from right to left. Blue dots inall of panels indicates the leftover target CCRF-CEM cells and red dotsshows effector cells by co-culture assay. All of incubation time were 16h and the ratio of effector T-cells: target cell is 5:1. All experimentswere performed in duplicate. (52B) Bar graph indicates the percent ofcell lysis by the CD5CAR NK-92 cells or CD5CAR NK^(45i)-92 cellscompared to the control GFP NK92 cells in co-culture assay withCCRF-CEM. Data are mean±S.D. Both of CD5CAR NK-cells and CD5CARNK^(45i)-92 cells shows near to 100% cell killing activity againstCD5-positive CCRF-CEM compared to control GFP NK-92 cells. These datasuggest that CD5CAR NK-cells and CD5CAR NK^(45i)-92 cells caneffectively lyse CCRF-CEM cells that express CD5 compared to GFP-controlNK-92 cells in vitro co-culture assay and prof that knockdown of CD45does not affect cell function for killing activity in NK-92 cells.

FIGS. 53A-53B. Organization of the CD45CAR construct and its expression.(53A) Schematic representation of the CD45CAR lentiviral vector. TheCD45CAR construct is a modularized signaling domain containing: a leadersequence, an anti-CD45scFv, a hinge domain (H), a transmembrane domain(TM), two co-stimulatory domains (CD28 and 4-1BB) that define theconstruct as a 3^(rd) generation CAR, and the intracellular signalingdomain CD3 zeta. (53B), HEK-293FT cells were transfected with lentiviralplasmids for GFP (lane 1) and CD45CAR (lane 2). 48 hours aftertransfection, supernatant was removed, and cells were also removed.Cells were lysed for Western blot and probe with mouse anti-human CD3zantibody.

FIG. 54 . Transduction of CD45CAR into NK^(45i)-92 cells and cellsorting of CD45CAR transduced cells. The expression levels of CD45CAR onNK^(45i)-92 were determined by flow cytometry analysis (circled in blueat middle panel) compared to NK^(45i)-92 cells (left panel) afterCD45CAR lentviruses were transduced into NK^(45i)-92 cells. CD45CARexpressed NK^(45i)-92 cells were sorted and CD45 expression levels oncell surface were determined by Flow cytometry analysis (right panel).About 87% of CD45CAR expression on cell surface was detected by flowcytometry analysis.

FIGS. 55A-55B. Co-culture assay with CCRF-CEM (target: T) and GFP NK-92or CD45CAR NK^(45i)-92 cells (effector: E) at 5:1 (E:T) ratio and 16hour incubation. (55A) Flow cytometry analysis of in co-culture withCCRF-CEM and control GFP transduced NK-92 cells (left panel) or CD45CARNK^(45i)-92 cells (right panel). Blue dots in all of panels indicatesthe leftover target CCRF-CEM cells and red dots shows effector NK-92cells by co-culture assay. All of incubation time were 16 h and theratio of effector T-cells: target cell is 5:1. All experiments wereperformed in duplicate. (55B) Bar graph indicates the percent of celllysis by CD45CAR NK^(45i)-92 cells compared to the control GFP NK92cells in co-culture assay with CCRF-CEM. Data are mean±S.D. CD45CARNK^(45i)-92 cells shows about 70% cell lysis against CCRF-CEM cellscompared to control GFP NK-92 cells. These data suggest that CD45CARNK^(45i)-92 cells effectively lyse CCRF-CEM cells that express CD45compared to GFP-control NK-92 cells in vitro co-culture assay.

FIGS. 56A-56C. Co-culture assay with Jurkat cells (target: T) andGFP-control or CD45CAR NK^(45i)-92 cells (effector: E) at 5:1 or 2:1(E:T) ratio and 6 hour incubation. (56A) Flow cytometry analysis wascarried out after Jurkat cells were stained by CMTMR cell tracker dye.These data show that Jurkat cells are CD45 positive (left panels) andmostly CD56 negative cells (right panel). (56B) Flow cytometry analysisof co-culture assay with Jurkat cells (target: T) and control or CD45CARNK^(45i)-92 cells (effector: E). The ratio of co-culture assay wasperformed in 5:1 or 2:1 (E: T). Left panels showed that in co-culturewith control GFP or CD45CAR/CD45KD NK-92 cells in 5:1 (E:T) ratio andright panels indicated that in co-culture with control GFP or CD45CARNK^(45i)-92 cells in 2:1 (E:T) ratio. Blue dots in panels indicate theleftover target Jurkat cells and red dots represent effector cells byco-culture assay. All of incubation time were 6 h. All experiments wereperformed in duplicate. (56C) Bar graph shows percent cell lysis byCD45CAR NK^(45i)-92 cells compared to control GFP NK92 cells at in 5:1or 2:1 (E: T) ratio. Data are mean±S.D. CD45CAR NK^(45i)-92 cells showsabout 60% cell lysis against Jurkat cells compared to control GFP NK-92cells in both conditions. This data suggests that CD45CAR NK^(45i)-92cells effectively lyse Jurkat cells that express CD45 on cell surfacecompared to GFP-control NK-92 cells in vitro co-culture assay.

FIGS. 57A-57J. Co-culture assay with GFP-NK-92 cells (target: T) andnon-transduced NK-92 cells or CD45CAR NK^(45i)-92 cells (effector: E) at5:1 or 2:1 (E:T) ratio, 6 hour incubation. (57A) Flow cytometry analysiswas carried out using GFP control NK-92 cells. These data proof that GFPcontrol NK-92 cells are about 99% GFP positive cells (green dots). (57B)Flow cytometry analysis of co-culture assay with GFP control NK-92 cells(target: T) and non-transduced or CD45CAR NK^(45i)-92 cells (effector:E). The ratio of co-culture assay was performed in 5:1 (57A) or 2:1(E:T) (57C). Left panels showed that in co-culture with non-transducedor CD45CAR NK^(45i)-92 cells in 5:1 (E:T) ratio and right panelsindicated that in co-culture with non-transduced or CD45CAR NK^(45i)-92cells in 2:1 (E:T) ratio. Green dots in panels indicate the leftovertarget GFP NK-92 cells and red dots represent effector cells byco-culture assay. All of incubation times were 6 h. All experiments wereperformed in duplicate. (57C) Bar graph shows percent cell lysis of GFPNK-92 cells by CD45CAR NK^(45i)-92 cells compared to non-transducedNK-92 cells at in 5:1 or 2:1 (E:T) ratio. Data are mean±S.D. CD45CARNK⁴⁵i-92 cells shows about 20% cell lysis in 2:1 (E:T) ratio and about55% cell lysis in 5:1 (E:T) ratio against GFP NK-92 cells compared tonon-transduced NK-92 cells. This data suggests that CD45CAR NK^(45i)-92cells effectively lyse GFP NK-92 cells that express CD45 on cell surfacecompared to non-transduced NK-92 cells in vitro co-culture assay. (57D).Transduction of CD45b-BB or CD45b-28 into NK^(45i)-92 cells and cellsorting of CD45b-BB or CD45b-28 transduced NK^(45i)-92 cells. Theco-stimulatory domain for CDb-BB is 4-1BB while co-stimulatory domainfor CD45b-28 is CD28. The expression levels of CD45b-BB or CD45b-28 onNK^(45i)-92 were determined by flow cytometry analysis (circled in blueat middle panel) compared to NK^(45i)-92 cells (left panel) afterCD45b-BB or CD45b-28 on lentviruses were transduced into NK^(45i)-92cells. CD45b-BB or CD45b-28 on expressed NK^(45i)-92 cells were sortedand CD45b-BB or CD45b-28 on expression levels on cell surface weredetermined by Flow cytometry analysis (right panel). About 74% ofCD45b-BB or 82% of CD45b-28 on expression on cell surface was detectedby flow cytometry analysis. (57E). Co-culture assay with REH cells(target: T) and GFP NK-92 cells, CD45CAR NK^(45i)-92 cells, CD45b-BBNK⁴⁵i-92 cells or CD45b-28 NK^(45i)-92 cells at 5:1 (E:T) ratio and 20hour incubation. Upper, Flow cytometry analysis of CREH cells only (leftpanel), in co-culture with with REH cells and control GFP transducedNK-92 cells (2^(nd) left panel), CD45CAR NK^(45i)-92 cells (middlepanel), CD45b-BB NK^(45i)-92 cells (4^(th) from left panel) or CD45b-28NK^(45i)-92 cells (right panel). Blue dots in all of panels indicatesthe leftover target REH cells and red dots shows effector GFP orCARs-NK-92 cells by co-culture assay. All of incubation time were 20hand the ratio of effector NK-cells: target cell is 5:1. All experimentswere performed in duplicate. Lower, Bar graph indicates the percent ofcell lysis by CD45CAR NK^(45i)-92 cells, CD45b-BB NK^(45i)-92 cells orCD45b-28 NK^(45i)-92 cells compared to the control GFP NK92 cells inco-culture assay with REH cells. Data are mean±S.D. CD45CAR NK^(45i)-92cells shows about 76% cell lysis, CD45b-BB NK^(45i)-92 cells shows about79% cell lysis and CD45b-28 NK^(45i)-92 shows 100% cell lysis againstREH cells compared to control GFP NK-92 cells. These data suggest thatthese 3 of CD45CARs NK^(45i)-92 cells effectively lyse REH cells whichcharacterized as B-cells expressing CD45 compared to GFP-control NK-92cells in vitro co-culture assay. Co-culture assay with U937 cells(target: T) and GFP NK-92 cells or CD45b-28 NK^(45i)-92 cells. at 2:1(E:T) ratio for 20 hours. (57FA), Flow cytometry analysis of U937 cells(monocytic leukemia cell line) only (left panel), in co-culture withU937 cells and control GFP transduced NK-92 cells (middle panel) orCD45b-28 NK^(45i)-92 cells (right panel). Blue dots in all of panelsindicates the leftover target U937 cells and red dots shows effector GFPor CD45b-28 NK^(45i)-92 cells by co-culture assay. All of incubationtime were 6h and the ratio of effector NK-cells: target cell is 2:1.(57FB), Bar graph indicates the percent of cell lysis by CD45b-28NK^(45i)-92 cells compared to the control GFP NK92 cells in co-cultureassay with U937 cells. CD45b-28 NK^(45i)-92 shows about 81% cell lysisagainst U937 cells compared to control GFP NK-92 cells.

Co-culture assay with MOLM-13 cells (target: T) and GFP NK-92 cells orCD45b-28 NK^(45i)-92 cells at 5:1 (E:T) ratio for 20 hours. (57GA), Flowcytometry analysis of MOLM13 cells (monocytic leukemic cell line) only(left panel), in co-culture with Molm13 cells and control GFP transducedNK-92 cells (middle panel) or CD45b-28 NK^(45i)-92 cells (right panel).Blue dots in all of panels indicates the leftover target MOLM13 cellsand red dots shows effector GFP or CD45b-28 NK^(45i)-92 cells byco-culture assay. All of incubation time were 20h and the ratio ofeffector NK-cells: target cell is 5:1. (57 GB), Bar graph indicates thepercent of cell lysis by CD45b-28 NK^(45i)-92 cells compared to thecontrol GFP NK92 cells in co-culture assay with MOLM13 cells. CD45b-28NK^(45i)-92 shows about 91.6% cell lysis against Molm13 cells comparedto control GFP NK-92 cells. Co-culture assay with Jeko-1 cells (target:T) and GFP NK-92 cells or CD45b-28 NK^(45i)-92 cells at 2:1 (E:T) ratiofor 6 hours. (57HA), Flow cytometry analysis of Jeko-1 cells (T cellacute lymphoblastic cell line) only (left panel), in co-culture withJeko-1 cells and control GFP transduced NK-92 cells (middle panel) orCD45b-28 NK^(45i)-92 cells (right panel). Blue dots in all of panels theleftover target Jeko-1 cells and red dots shows effector GFP or CD45b-28NK^(45i)-92 cells by co-culture assay. All of incubation time were 6hand the ratio of effector NK-cells: target cell is 2:1. (57HB). Bargraph indicates the percent of cell lysis by CD45b-28 NK^(45i)-92 cellscompared to the control GFP NK92 cells in co-culture assay with Jeko-1cells. CD45b-28 NK^(45i)-92 shows about 44.6% cell lysis against Jeko-1cells compared to control GFP NK-92 cells. Co-culture assay with SP53cells (target: T) and GFP NK-92 cells or CD45b-28 NK^(45i)-92 cells at2:1 (E:T) ratio for 6 hour incubation. (57IA), Flow cytometry analysisof SP53 cells (mantle cell lymphoma cell line) only (left panel), inco-culture with Jeko-1 cells and control GFP transduced NK-92 cells(middle panel) or CD45b-28 NK^(45i)-92 cells (right panel). Blue dots inall of panels indicates the leftover target SP53 cells and red dotsshows effector GFP or CD45b-28 NK^(45i)-92 cells by co-culture assay.All of incubation time were 6h and the ratio of effector NK-cells:target cell is 2:1. (57IB), Bar graph indicates the percent of celllysis by CD45b-28 NK^(45i)-92 cells compared to the control GFP NK92cells in co-culture assay with SP53 cells. CD45b-28 NK^(45i)-92 showsabout 45% cell lysis against SP53 cells compared to control GFP NK-92cells.(57J). Elimination of CD34(+) umbilical chord blood stem cells in 48 hrco-culture. CD34(+) stem cells derived from human umbilical cord bloodwere co-cultured with either Control or CD45b-28 CAR NK cells for 48 hrprior to labeling at a low ratio of 2:1 (effective: target). About 96%of CD34(+) cells were eliminated comparing to the control.

FIGS. 58A-58C. A Link by P2A schematic showing both cCAR-T and 4-1BBL orIL-15/IL-15sushi in a single construct. (58A) The construct consists ofa SFFV promoter driving the expression of CAR and an enhancer, 4-1BBL.Upon cleavage of the linkers, the CD45 CAR (or CD45b CAR) and 4-1BBL orIL-15/IL-15sush split and engage upon targets expressing CD45. CD45 CART cells received not only costimulation through the CD28 but also 4-1BBligand (4-1BBL or CD137L) or IL-15/IL-15sushi. The CD3-zeta signalingdomain completes the assembly of this CAR-T. (58B). Surface CD45b CARexpression levels on CD45b-28-2G-4-1BBL CAR transduced NK^(45i)-92 cellswere determined using flow cytometry analysis. Left panel (NK92 cells)and middle panel (GFP-NK92) indicated negative control and right panelshowed the surface expression of CD45b CAR which was labeled using goatanti-mouse F(AB′)2-PE against ScFv region (circled in blue).Transducedcells expressed 86.99% of CD45b-CAR on the cell surface. (58C). SurfaceCD45b CAR expression levels on CD45b-28-2G-IL15/IL-15sushi CARtransduced NK45i-92 cells were determined using flow cytometry analysis.Left panel (NK92 cells) and middle panel (GFP-NK92) indicated negativecontrol and right and right panel showed the surface expression of CD45bCAR which was labeled using goat anti-mouse F(AB′)2-PE against ScFvregion (circled in blue). CD45b-28-2G IL15RA virus transduced cellsexpressed 55.96% of CD45b-CAR on cells surface compared to negativecontrol cells.

FIGS. 59A-59B. Schematic diagram to elucidate the construct and itsexpression in T or NK cells. (59A) a combination of CAR, (thirdgeneration), and IL-15/sushi domain of the IL-15 alpha receptor, isassembled on an expression vector and their expression is driven by theSFFV promoter. CAR with IL-15/sushi is linked with the P2A self-cleavingsequence. The IL-15/sushi portion is composed of IL-2 signal peptidefused to IL-15 and linked to sushi domain via a 26-amino acidpoly-proline linker. (59B) CAR and IL-15/sushi are present on the T orNK cells. Surface CD45b CAR expression levels onCD45b-28-2G-IL-15/IL-15sushi CAR transduced NK^(45i)-92 cells weredetermined using flow cytometry analysis. Left panel (NK92 cells) andmiddle panel (GFP-NK92) indicated negative control and right and rightpanel showed the surface expression of CD45b CAR which was labeled usinggoat anti-mouse F(AB′)2-PE against ScFv region (circled in blue).CD45b-28-2G IL-15/IL-15sushi virus transduced cells expressed 55.96% ofCD45b-CAR on cells surface compared to negative control cells.CD45b-28-2G-IL-15/IL-15sushi NK cells showed a robust functionalactivity.

FIGS. 60A-60B. CD4IL-15/IL-15sushi expression. (60A) HEK-293FT cellswere transfected with lentiviral plasmids for GFP (lane 1) andCD4IL-15/IL-15sushi CAR (lane 2). 48 hours after transfection,supernatant was removed, and cells were also removed for a Western blotwith mouse anti-human CD3z antibody. (60B) HEK-293 cells were transducedwith either GFP (left) or CD4IL-15/IL-15sushi-CAR (right) viralsupernatant from transfected HEK-293FT cells. After 3 days incubation,cells were harvested, stained with goat-anti-mouse F(Ab′)2 and analyzedby flow cytometry.

FIG. 61 . Transduction of NK cells with CD4IL-15/IL-15sushi CAR. NK-92cells were transduced with either GFP (left) or CD4 IL-15/IL-15sushi CAR(right) viral supernatant from transfected HEK-293FT cells. A secondtransduction was performed 24 hours after the first. 24 hours after thesecond transduction, cells were harvested, washed and moved to tissueculture plates with fresh media and IL-2. After 3 days incubation, cellswere harvested and stained with goat-anti-mouse F(Ab′)2 antibody or goatIgG (control) at 1:250 for 30 minutes. Cells were washed and stainedwith streptavidin-PE conjugate at 1:500, washed, suspended in 2%formalin, and analyzed by flow cytometry.

FIG. 62 . Transduction of T cells with CD4IL15RACAR. Left is the Westernblot. HEK-293FT cells were transfected with lentiviral plasmids for GFP(lane 1) and CD4IL15RA-CAR (lane 2). 48 hours after transfection,supernatant was removed, and cells were also collected for a Westernblot with mouse anti-human CD3zeta antibody. Right is CD4IL15RACARexpression. Activated T cells from cord blood buffy coat were transducedwith either GFP (left) or concentrated CD4IL15RACAR (right) viralsupernatant from transfected HEK-293FT cells. A second transduction wasperformed 24 hours after the first. 24 hours after the secondtransduction, cells were harvested, washed and moved to tissue cultureplates with fresh media and IL-2. After 3 days incubation, cells wereharvested and stained with goat-anti-mouse F(Ab′) transduced with eitherGFP (left) or CD4IL15RA CAR (right). Cells were washed and stained withstreptavidin-PE conjugate at 1:500, washed, suspended in 2% formalin,and analyzed by flow cytometry.

FIGS. 63A-63B. CD4CAR NK-92 cells and CD4IL-15/IL-15sushi CAR NK-92cells eliminate KARPAS 299 T leukemic cells in co-culture. (63A) NK-92cells transduced with either GFP control (upper right), CD4CAR (lowerleft), or CD4IL-15/IL-15sushi (lower right) lentiviral supernatant wereincubated with KARPAS 299 cells at a ratio of 5:1. After 4 hoursco-culture, cells were stained with mouse-anti-human CD4 (APC) and CD3(PerCp) antibodies and analyzed by flow cytometry (N=2). The upper leftpanel shows labeled Karpas 299 cells alone. The percentage of targetcells lysed is shown in the graph (63B).

FIG. 64 . CD4CAR NK-92 cells and CD4IL-15/IL-15sushi CAR NK-92 cellseliminate MOLT4 T leukemic cells in co-culture. NK-92 cells transducedwith either GFP control (left), CD4CAR (center), or CD4IL-15/IL-15sushi(second from right) lentiviral supernatant were incubated with MOLT4cells at effector:target ratios of 1:1 or 2:1. After overnightco-culture, cells were stained with mouse-anti-human CD4 (APC) and CD56(PerCp) antibodies and analyzed by flow cytometry (N=2). The upper rightpanel shows labeled MOLT4 cells alone. The percentage of target cellslysed is shown in the graph.

FIGS. 65A-65D. CD4CAR and CD4IL-15/IL-15sushi CAR T cells demonstrateanti-leukemic effects in vivo. NSG mice were sublethally irradiated andintravenously (tail vein) injected the following day withluciferase-expressing MOLM13 cells to induce measurable tumor formation(65A). MOLM-13 cells are nearly 100% CD4+. After 3 days, the mice wereintravenously injected with one course of 8×10⁶ CD4CAR, orCD4IL-15/IL-15sushi CAR T cells, or vector control T control cells. Ondays 3, 6, 9, and 11, mice were injected subcutaneously with RediJectD-Luciferin and subjected to IVIS imaging (65B). (65C) Average lightintensity measured for the CD4CAR and CD4IL-15/IL-15sushi CAR T injectedmice was compared to that of vector control T injected mice, andcorrelated with remaining tumor burden to determine a percent lysis.(65D) Percent survival of mice was measured and compared between thethree groups.

FIG. 66A-66C. CD4IL15/IL-15sushi CAR NK cells demonstrate robustanti-leukemic activity under stressful condition in vivo. NSG mice weresublethally irradiated and intravenously (tail vein) injected thefollowing day with luciferase-expressing Jurkat cells to inducemeasurable tumor formation (66A). Jurkat cells are less than 60% CD4+.After 3 days, the mice were intravenously injected with one course of8×10⁶ CD4CAR, or CD4IL-15/IL-15sushi CAR NK cells, or vector control NKcells. On days 3, 7, 10, and 14, mice were injected subcutaneously withRediJect D-Luciferin and subjected to IVIS imaging (66B). (66C and 66D).Average light intensity measured for the CD4CAR and CD4IL-15/IL-15sushiNK injected mice was compared to that of vector control NK injectedmice, and correlated with remaining tumor burden to determine a percentlysis.

FIGS. 67A-B. Repeat of the in vivo experiment demonstrating robust lysisof Jurkat tumor cells by CD4I-15/IL-15sushi CAR NK cells showing similarresults to those described in FIG. 66 .

FIGS. 68A-68B. Effect of secreted IL-15/IL-15sushi on CAR andnon-transduced neighboring cells. NK-92 cells stably expressing eitherCD4CAR or CD4IL15RA were mixed in a 50:50 ratio with NK-92 cells stablyexpressing GFP. These cells were co-cultured either with IL-2 added orno IL-2. (68A) Photographs taken on a fluorescent microscope at 20× onDay 0 (start of co-culture) and Day 7, without the addition of IL-2.(68B) Total cell counts calculated throughout the experiment (up to Day14) for NK-92 cells co-cultured with or without IL-2.

FIG. 69 . Comparing the effect of secreted IL-15 and IL-15sushi on NK-92cell growth. CD4IL-15/IL-15sushi, CD4 IL-15, and control transducedNK-92 cells were cultured from 250,000 cells in regular NK cell mediabut in the absence of IL-2 for up to 6 days. Both transduced cells had10% surface CAR expression, while CD4IL15-IL15sushi transduced NK-92cells were able to expand at a rate approximately 3-fold higher than theCD4 IL-15 transduced NK-92 cells on day 6. On day 4, the growth rate ofCD4 IL-15 transduced NK-92 cells were slightly higher than the Control,but significantly below the CD4 IL-15/IL15sushi transduced NK-92 cells.This study pin-points the importance of co-expression functional complexof IL-15/IL-15sushi in promoting NK-92 cell growth.

FIG. 70 . A schematic showing the Treg CAR T construct targeting Tregs.The construct consists of a SFFV promoter driving the expression of twounits of chimeric antigen receptors linked by a P2A peptide. Each unitcontains a CD45 leader peptide sequence (signal peptide). Upon cleavageof the linker, two units of peptide are divided and engage upon targetsexpressing CD4 and CD25. The CD4 chimeric antigen receptor polypeptideunit comprises a signal peptide, a CD4 antigen recognition domain, ahinge region, a transmembrane domain and CD3 zeta chain; CD25 chimericantigen receptor polypeptide unit comprises a signal peptide, a CD25antigen recognition domain, a hinge region, a transmembrane domain, aco-stimulatory domain (s). The Treg CAR can potentiate the lysisactivity of a cell co-expressing CD4 and CD25 while minimizing a cellbearing CD4 or CD25 antigen.

FIGS. 71A-71B. Characterization of the CD4 zeta CD25 CAR. (71A) The CD4zeta CD25 CAR was transduced into T-cells via viral incubation for 48hours and stained with F(ab)′ antibody to assay CAR surface expression.Encircled populations represent transduced cells. (71B) The C4-25z CARwas characterized using CD4 and CD25 antibodies to validate theconstruct function. Two most relevant populations are encircled:CD4+CD25+ and CD4⁻ CD25⁺. The depletion of the double positivepopulation and other phenotype groups are summarized in the bar graphadjacent.

FIG. 72 . CD4zetaCD25 CAR T cells target cells mainly co-expressing CD4and CD25. 3 days after activation, PMBC buffy coat T cells transducedwith either control vector (left), CD4CAR (middle) or CD4zetaCD25(right)lentiviral supernatant were harvested and incubated with mouseanti-human CD25-PE and mouse anti-human CD4-APC for 30 minutes. Cellswere washed and suspended in 2% formalin, and analyzed by flowcytometry.

FIGS. 73A-73D. A schematic showing the CD52-52 construct. The constructconsists of a SFFV promoter driving the co-expression of CD5CAR and CD52surface antigen. Upon cleavage of the linker of P2A. (73A) The CD5chimeric antigen receptor polypeptide unit comprises a signal peptide, aCD5 antigen recognition domain, a hinge region, a transmembrane domainand CD3 zeta chain; CD5 peptide comprises a signal peptide, a CD52antigen recognition domain, a hinge region, a transmembrane domain(derived from CD28). (73B). Experimental design to determine depletionof CD5CAR-52 T cells in blood. CD5CAR-52 T cells (5×10⁶ cells) wereinjected intravenously into each NSG mouse after sublethallyirradiation. After ˜24 h later, PBS or 0.1 mg/kg of CAMPATH was injectedvia I.P. (intraperitoneal injection). N=3. After 6 h and 24 h later,peripheral blood was collected from each mouse and labeled using CD3 andCD45 antibodies to determine the depletion of CAR-T cells as acute phaseresponse by CAMPATH treatment. After 5 days later, whole blood wascollected from each mouse and labeled using CD3 and CD45 antibodies todetermine the persistency of CAR-T cells as well. CAR-T-cells weredetermined using Flow cytometry analysis. (73C). Depletion of CD5CAR-52T in peripheral blood after 6 h and 24 h later with or without CAMPATHtreatment. Flow cytometry analysis shows persistence of CD5CAR-52T-cells (Blue dots) in peripheral blood of mouse with or without CAMPATHtreatment. Blood samples were labeled with CD3 and CD45 antibodies todetect CD5CAR-52 T-cells. Blood samples from un-infused CAR-T cells(left panels) did not show CD3 and CD45 positive cells (negativecontrol). 0.1 mg/kg of CAMPATH injected mice indicate elimination ofCD5CAR-52 T-cells at 6h (middle panels) and 24 h (right panels) latercompared to CAMPATH untreated mouse at 6h (second panels from left) and24 h (second panels from right) in blood samples. N=3. These resultssuggest that CAMPAT treatment can delete CAR-T cells from blood duringshort time. (73D). Depletion of CD5CAR-52 T in whole blood after 5 dayslater with or without CAMPATH treatment. Flow cytometry analysis showspersistence of CD5CAR-52 T-cells (Blue dots) in whole blood samples frommouse with or without CAMPATH treatment. Blood samples were labeled withCD3 and CD45 antibodies to detect CD5CAR-52 T-cells persistence. Bloodsamples from uninfused CAR-T cells (left panel) did not show CD3 andCD45 positive cells (negative control). 0.1 mg/kg of CAMPATH treatedmice eliminate CD5CAR-52 T-cells (right panels) compared to CAMPATHuninjected mouse (middle panels) after 5 days later in whole bloodsamples. These results also suppose CAMPAT treatment can delete CAR-Tcells from blood.

FIG. 74 . HEK 293 cells were transduced with either EF1-GFP or SFFV-GFPviral supernatant, using the volumes indicated, in DMEM with 10% FBS ina 6 well tissue culture plate. Culture media was changed the followingmorning. Forty-eight hours later, transduced cells were visualized on anEVOS fluorescent microscope using GFP at 10×.

FIG. 75 . HEK 293 cells transduced with either EF1-GFP or SFFV-GFP viralsupernatant, using the volumes from the previous figure, weretrypsinized, suspended in formalin, and subjected to flow cytometryanalysis, using the FITC channel to determine the percentage of GFP+cells.

FIGS. 76A-76B. Activated cord blood buffy coat T cells transduced witheither EF1-GFP or SFFV-GFP viral supernatant, with either low or highamounts of viral supernatant, were trypsinized, suspended in formalin,and subjected to flow cytometry analysis, using the FITC channel todetermine the percentage of GFP+ cells, 7, 14, 21 and 28 days aftertransduction. (76A) Percent GFP+ T cells for cells transduced witheither low or high amounts of supernatant. (76B) Percent of GFP+ T cellstransduced with the high amount of EF1-GFP supernatant, relative to thepercent GFP+ cells in the T cells transduced with the lower amount ofSFFV-GFP supernatant. (50 μL of SFFV-GFP and 1 mL of EF1-GFP supernatantwas used). (N=2).

FIG. 77 . Ligand receptor interactions in malignant plasma cells. TheAPRIL ligand binds TAC1 or BCMA. The BAFF ligand binds TAC1, BCMA, orBAFF-R.

FIG. 78 . Steps for elimination of tumor by CAR co-expressing secretoryIL-15/IL-15sushi. I, tumor and its microenviroment. Macrophages, Tcells, dendritic cells and NK cells are immune response cells againsttumor in the tumor microenvironment and they secrete a low level ofendogenous IL-15, which is unstable, which complexes with the solubleextracellular domain of IL-15RA. The complex forms a more stablemolecule, which greatly enhances immune cell survival and expansion. Inthe tumor microenvironment, cancer cells express programmed death ligand1 (PD-L1) as a transmembrane protein that has been considered to play amajor role in suppressing the immune system during particular eventsincluding cancer. PD-L1 binds to its receptor, PD-1, found on activatedT cells, B cells, and myeloid cells, to suppress these cell immuneactivities. II, CAR T or NK cells targeting tumor cells, could be acarrier to deliver an enhancer to the tumor microenvironment. CAR T orNK cells are engineered to co-express a secretory fusion protein,IL-15/IL-15sushi fusion. III, Engineered CAR T or NK cells bind totargeted tumor cells (either subset or all cells). IV, Engineered CAR Tor NK cells in tumor microenvironment target tumor cells, binding to theCAR targeting antigen, and triggering lysis of tumor cells and massivesecretion of soluble IL-15/IL-15sushi fusion from the expansion of CAR Tor NK cells. The soluble IL-15/IL-15sushi fusion are stable andfunctions as an unexpected and powerful immunomodulatory for CAR T/NKcells and their neighbor tumor immune response cells. The secretedIL-15/IL-15sushi protein would be involved in trafficking of other Tcells, dendritic cells, macrophages and NK cells to the tumormicroenvironment, which then also: 1) lyse the tumor cells bysupplementing the defect that CAR T or NK cells are unable to eliminatenon-targeting cancer cells; 2) enhance CAR T/NK cell persistency andanti-tumor activity. The overexpression of IL-15/IL-15sushi overwhelmsthe PD-L1 ability to suppress the immune response. Preferably, this CARtherapy could be used synergistically with administration of acheckpoint blockage including, but not limited to PD-L1, CTLA-4inhibitor for even greater efficacy.

FIG. 79 . Surface markers during B cell and plasma cell development areshown. Both BAFF and APRIL binds to receptors, BCMA and TACI. BAFF alsobinds to BAFF-R receptor.

FIG. 80 . Protein sequence alignment of IL-2 signal peptide amongdifferent species.

FIG. 81 . Protein sequence alignment of BAFF extracellular domain amongdifferent species.

DETAILED DESCRIPTION

The disclosure provides chimeric antigen receptor (CAR) compositions,methods of making and using thereof.

A chimeric antigen receptor (CAR) polypeptide includes a signal peptide,an antigen recognition domain, a hinge region, a transmembrane domain,at least one co-stimulatory domain, and a signaling domain.

First-generation CARs include CD3z as an intracellular signaling domain,whereas second-generation CARs include at least one singleco-stimulatory domain derived from various proteins. Examples ofco-stimulatory domains include, but are not limited to, CD28, CD2, 4-1BB(CD137, also referred to as “4-BB”), and OX-40 (CD124). Third generationCARs include two co-stimulatory domains, such as, but not limited to,CD28, 4-1BB, CD134 (OX-40), CD2, and/or CD137 (4-1BB).

As used herein, the terms “peptide,” “polypeptide,” and “protein” areused interchangeably, and refer to a compound having amino acid residuescovalently linked by peptide bonds. A protein or peptide must contain atleast two amino acids, and no limitation is placed on the maximum numberof amino acids that can be included in a protein's or peptide'ssequence. Polypeptides include any peptide or protein having two or moreamino acids joined to each other by peptide bonds. As used herein, theterm refers to both short chains, which also commonly are referred to inthe art as peptides, oligopeptides, and oligomers, for example, and tolonger chains, which generally are referred to in the art as proteins,of which there are many types. “Polypeptides” include, for example,biologically active fragments, substantially homologous polypeptides,oligopeptides, homodimers, heterodimers, variants of polypeptides,modified polypeptides, derivatives, analogs, and fusion proteins, amongothers. The polypeptides include natural peptides, recombinant peptides,synthetic peptides, or a combination thereof.

A “signal peptide” includes a peptide sequence that directs thetransport and localization of the peptide and any attached polypeptidewithin a cell, e.g. to a certain cell organelle (such as the endoplasmicreticulum) and/or the cell surface. As used herein, “signal peptide” and“leader sequence” are used interchangeably.

The signal peptide is a peptide of any secreted or transmembrane proteinthat directs the transport of the polypeptide of the disclosure to thecell membrane and cell surface, and provides correct localization of thepolypeptide of the present disclosure. In particular, the signal peptideof the present disclosure directs the polypeptide of the presentdisclosure to the cellular membrane, wherein the extracellular portionof the polypeptide is displayed on the cell surface, the transmembraneportion spans the plasma membrane, and the active domain is in thecytoplasmic portion, or interior of the cell.

In one embodiment, the signal peptide is cleaved after passage throughthe endoplasmic reticulum (ER), i.e. is a cleavable signal peptide. Inan embodiment, the signal peptide is human protein of type I, II, III,or IV. In an embodiment, the signal peptide includes an immunoglobulinheavy chain signal peptide.

In one embodiment, the signal peptide includes the signal peptide fromhuman CD45. (UniProtKB/Swiss-Prot Accession Number P08575). The CD45signal peptide is 23 amino acids in length (MYLWLKLLAFGFAFLDTEVFVTG). Insome embodiments, the signal peptide may be a functional fragment of theCD45 signal peptide. A functional fragment includes a fragment of atleast 10 amino acids of the CD45 signal peptide that directs theappended polypeptide to the cell membrane and cell surface. Examples offragments of the human CD45 signal peptide include: MYLWLKLLAFG,FAFLDTEVFVTG, and LKLLAFGFAFLDTE.

Functional equivalents of the human CD45 signal peptide have also beencontemplated. As used herein, “functional equivalents” are to beunderstood as mutants that exhibit, in at least one of theabovementioned sequence positions, an amino acid substitution other thanthe one mentioned specifically, but still lead to a mutant which showthe same or similar properties with respect to the wild-type CD45 signalpeptide. Functional equivalents include polypeptides having at least80%, at least 85%, at least 90%, or at least 95% identity to the humanCD45 signal peptide, functional fragments thereof, or functionalequivalents thereof.

Functional equivalents also include CD45 signal peptides from homologousproteins from other species. Examples of these signal peptides includesignal peptide from mouse CD45 (MGLWLKLLAFGFALLDTEVFVTG); signal peptidefrom rat CD45 (MYLWLKLLAFSLALLGPEVFVTG); signal peptide from sheep CD45(MTMYLWLKLLAFGFAFLDTAVSVAG); signal peptide from chimpanzee CD45(MYLWLKLLAFGFAFLDTEVFVTG); and signal peptide from monkey CD45(MTMYLWLKLLAFGFAFLDTEVFVAG.

In another embodiment, the signal peptide includes the followingsequence: MX^(I)LWLKLLAF X²X³AX⁴LX⁵X⁶X⁷VX⁸ VX⁹G; wherein X¹, X², X³, X⁴,X⁵, X⁶, X⁷, X⁸, and X⁹ are independently Y, G, S, F, L, D, P, T, E, orA. In one embodiment, X¹ is Y or G; X² is G or S; X³ and X⁴ areindependently F or L; X⁵ is D or G; X⁶ is P or T; X⁷ is E or A; X⁸ is For S; and X⁹ is A or T.

In one embodiment, the signal peptide includes the signal peptide fromhuman CD8a (MALPVTALLLPLALLLHAARP). In some embodiments, the signalpeptide may be a functional fragment of the CD8a signal peptide. Afunctional fragment includes a fragment of at least 10 amino acids ofthe CD8a signal peptide that directs the appended polypeptide to thecell membrane and cell surface. Examples of fragments of the human CD8asignal peptide include: MALPVTALLLPLALLLHAA, MALPVTALLLP, PVTALLLPLALL,and LLLPLALLLHAARP.

In another embodiment, the signal peptide includes the signal peptidefrom human CD8b (MRPRLWLLLAAQLTVLHGNSV). In some embodiments, the signalpeptide may be a functional fragment of the CD8b signal peptide. Afunctional fragment includes a fragment of at least 10 amino acids ofthe CD8b signal peptide that directs the appended polypeptide to thecell membrane and cell surface. Examples of fragments of the human CD8bsignal peptide include: MRPRLWLLLAAQ, RLWLLLAAQLTVLHG, andLWLLLAAQLTVLHGNSV.

Functional equivalents of the human CD8a or CD8b signal peptide havealso been contemplated. As used herein, “functional equivalents” are tobe understood as mutants which exhibit, in at least one of theabovementioned sequence positions, an amino acid substitution other thanthe one mentioned specifically, but still lead to a mutant which showthe same or similar properties with respect to the wild-type CD8a orCD8b signal peptide. Functional equivalents include polypeptides havingat least 80%, at least 85%, at least 90%, or at least 95% identity tothe human CD8 signal peptide, functional fragments thereof, orfunctional equivalents thereof.

Functional equivalents also include CD8a and CD8b signal peptides fromhomologous proteins from other species.

In one embodiment, the signal peptide includes the signal peptide fromhuman IL-2. The IL-2 signal peptide is 23 amino acids in length(MYRMQLLSCIALSLALVTNS). In some embodiments, the signal peptide may be afunctional fragment of the IL-2 signal peptide. A functional fragmentincludes a fragment of at least 10 amino acids of the IL-2 signalpeptide that directs the appended polypeptide to the cell membrane andcell surface. Examples of fragments of the human IL-2 signal peptideinclude: MYRMQLLSCIAL, QLLSCIALSLAL, and SCIALSLALVTNS.

Functional equivalents of the human IL-2 signal peptide have also beencontemplated. As used herein, “functional equivalents” are to beunderstood as mutants which exhibit, in at least one of theabovementioned sequence positions, an amino acid substitution other thanthe one mentioned specifically, but still lead to a mutant which showthe same or similar properties with respect to the wild-type IL-2 signalpeptide. Functional equivalents include polypeptides having at least80%, at least 85%, at least 90%, or at least 95% identity to the humanIL-2 signal peptide, functional fragments thereof, or functionalequivalents thereof.

Functional equivalents also include IL-2 signal peptides from homologousproteins from other species. See for example FIG. 80 .

In another embodiment, the signal peptide includes the followingsequence: MYX¹X²QLX³SCX⁴X⁵LX⁶LX⁷LX⁸X⁹X¹⁰X¹¹; wherein X¹, X², X³, X⁴, X⁵,X⁶, X⁷, X⁸, X⁹, X¹⁰, and X¹¹are independently R, K, S, M, I, V, L, A, I,T, N, S, or G. In one embodiment, X¹ is R, K, or S; X² is M, I, or V; X³is L or A; X⁴ and X⁵ are independently I, A, V, or T; X⁶ is S or T; X⁷is A or V; X⁸, X⁹, X¹⁰, and X¹¹ are independently V, L, T, A, N, S, orG.

The “antigen recognition domain” includes a polypeptide that isselective for or targets an antigen, receptor, peptide ligand, orprotein ligand of the target; or a polypeptide of the target.

The antigen recognition domain may be obtained from any of the widevariety of extracellular domains or secreted proteins associated withligand binding and/or signal transduction. The antigen recognitiondomain may include a portion of Ig heavy chain linked with a portion ofIg light chain, constituting a single chain fragment variable (scFv)that binds specifically to a target antigen. The antibody may bemonoclonal or polyclonal antibody or may be of any type that bindsspecifically to the target antigen. In another embodiment, the antigenrecognition domain can be a receptor or ligand. In particularembodiments, the target antigen is specific for a specific diseasecondition and the disease condition may be of any kind as long as it hasa cell surface antigen, which may be recognized by at least one of thechimeric receptor construct present in the compound CAR architecture. Ina specific embodiment, the chimeric receptor may be for any cancer forwhich a specific monoclonal or polyclonal antibody exists or is capableof being generated. In particular, cancers such as neuroblastoma, smallcell lung cancer, melanoma, ovarian cancer, renal cell carcinoma, coloncancer, Hodgkin's lymphoma, and childhood acute lymphoblastic leukemiahave antigens specific for the chimeric receptors.

In some embodiments, antigen recognition domain can be non-antibodyprotein scaffolds, such as but not limited to, centyrins, non-antibodyprotein scaffolds that can be engineered to bind a variety of specifictargets with high affinity. Centyrins are scaffold proteins based onhuman consensus tenascin FN3 domain, are usually smaller than scFvmolecules CAR molecules.

The target specific antigen recognition domain preferably includes anantigen binding domain derived from an antibody against an antigen ofthe target, or a peptide binding an antigen of the target, or a peptideor protein binding an antibody that binds an antigen of the target, or apeptide or protein ligand (including but not limited to a growth factor,a cytokine, or a hormone) binding a receptor on the target, or a domainderived from a receptor (including but not limited to a growth factorreceptor, a cytokine receptor or a hormone receptor) binding a peptideor protein ligand on the target.

In one embodiment, the antigen recognition domain includes the bindingportion or variable region of a monoclonal or polyclonal antibodydirected against (selective for) the target.

In another embodiment, the antigen recognition domain includes Camelidsingle domain antibody, or portions thereof. In one embodiment, Camelidsingle-domain antibodies include heavy-chain antibodies found incamelids, or VHH antibody. A VHH antibody of camelid (for example camel,dromedary, llama, and alpaca) refers to a variable fragment of a camelidsingle-chain antibody (See Nguyen et al, 2001; Muyldermans, 2001), andalso includes an isolated VHH antibody of camelid, a recombinant VHHantibody of camelid, or a synthetic VHH antibody of camelid.

In another embodiment, the antigen recognition domain includes ligandsthat engage their cognate receptor. By way of example, APRIL is a ligandthat binds the TAC1 receptor or the BCMA receptor. In accordance withthe present disclosure, the antigen recognition domain includes APRIL,or a fragment thereof. By way of further example, BAFF is a ligand thatbinds the BAFF-R receptor or the BCMA receptor. In accordance with thepresent disclosure, the antigen recognition domain includes BAFF, or afragment thereof. In another embodiment, the antigen recognition domainis humanized.

It is understood that the antigen recognition domain may include somevariability within its sequence and still be selective for the targetsdisclosed herein. Therefore, it is contemplated that the polypeptide ofthe antigen recognition domain may be at least 95%, at least 90%, atleast 80%, or at least 70% identical to the antigen recognition domainpolypeptide disclosed herein and still be selective for the targetsdescribed herein and be within the scope of the disclosure.

The target includes interleukin 6 receptor, NY-ESO-1, alpha fetoprotein(AFP), glypican-3 (GPC3), BCMA, BAFF-R, TACI, LeY, CD13, CD14, CD15CD19, CD20, CD22, CD33, CD41, CD61, CD64, CD68, CD117, CD123, CD138,CD267, CD269, CD38, Flt3 receptor, CS1, CD45, ROR1, PSMA, MAGE A3,Glycolipid, glypican 3, F77, GD-2, WT1, CEA, HER-2/neu, MAGE-3, MAGE-4,MAGE-5, MAGE-6, alpha-fetoprotein, CA 19-9, CA 72-4, NY-ESO, FAP, ErbB,c-Met, MART-1, CD30, EGFRvIII, immunoglobin kappa and lambda, CD38,CD52, CD3, CD4, CD8, CD5, CD7, CD2, and CD138

In another embodiment, the target includes any portion interleukin 6receptor, NY-ESO-1, alpha fetoprotein (AFP), glypican-3 (GPC3), BCMA,BAFF-R, TACI, LeY, CD13, CD14, CD15 CD19, CD20, CD22, CD33, CD41, CD61,CD64, CD68, CD117, CD123, CD138, CD267, CD269, CD38, Flt3 receptor, CS1,CD45, TACI, ROR1, PSMA, MAGE A3, Glycolipid, glypican 3, F77, GD-2, WT1,CEA, HER-2/neu, MAGE-3, MAGE-4, MAGE-5, MAGE-6, alpha-fetoprotein, CA19-9, CA 72-4, NY-ESO, FAP, ErbB, c-Met, MART-1, CD30, EGFRvIII,immunoglobin kappa and lambda, CD38, CD52, CD3, CD4, CD8, CD5, CD7, CD2,and CD138.

In one embodiment, the target includes surface exposed portions ofinterleukin 6 receptor, NY-ESO-1, alpha fetoprotein (AFP), glypican-3(GPC3), BCMA, BAFF-R, TACI, LeY, CD13, CD14, CD15 CD19, CD20, CD22,CD33, CD41, CD61, CD64, CD68, CD117, CD123, CD138, CD267, CD269, CD38,Flt3 receptor, CS1, CD45, TACI, ROR1, PSMA, MAGE A3, Glycolipid,glypican 3, F77, GD-2, WT1, CEA, HER-2/neu, MAGE-3, MAGE-4, MAGE-5,MAGE-6, alpha-fetoprotein, CA 19-9, CA 72-4, NY-ESO, FAP, ErbB, c-Met,MART-1, CD30, EGFRvIII, immunoglobin kappa and lambda, CD38, CD52, CD3,CD4, CD8, CD5, CD7, CD2, and CD138 polypeptides.

For example, the target includes the surface exposed regions of BAFF, asshown in FIG. 81 . The target may include a portion of the surfaceexposed regions of BAFF. For example, portions of BAFF include residues1-200, 1-100, 50-150, or 100-200 of human BAFF.

In another embodiment, the target antigens include viral or fungalantigens, such as E6 and E7 from the human papillomavirus (HPV) or EBV(Epstein Barr virus) antigens; portions thereof; or surface exposedregions thereof.

In one embodiment, the TACI antigen recognition domain includes SEQ IDNO. 24.

In one embodiment, the BCMA antigen recognition domain includes SEQ IDNO. 25.

In one embodiment, the CS1 antigen recognition domain includes SEQ IDNO. 26.

In one embodiment, the BAFF-R antigen recognition domain includes SEQ IDNO. 27.

In one embodiment, the CD33 antigen recognition domain includes SEQ IDNO. 28.

In one embodiment, the CD123 antigen recognition domain includes SEQ IDNO. 29.

In one embodiment, the CD19 antigen recognition domain includes SEQ IDNO. 30.

In one embodiment, the CD20 antigen recognition domain includes SEQ IDNO. 31. In another embodiment, the CD20 antigen recognition domainincludes SEQ ID NO. 32.

In one embodiment, the CD22 antigen recognition domain includes SEQ IDNO. 33.

In on embodiment, the CD45 antigen recognition domain includes SEQ IDNO. 34.

In on embodiment, the CD4 antigen recognition domain includes SEQ ID NO.35

In on embodiment, the CD25 antigen recognition domain includes SEQ IDNO. 36

The hinge region is a sequence positioned between for example,including, but not limited to, the chimeric antigen receptor, and atleast one co-stimulatory domain and a signaling domain. The hingesequence may be obtained including, for example, from any suitablesequence from any genus, including human or a part thereof. Such hingeregions are known in the art. In one embodiment, the hinge regionincludes the hinge region of a human protein including CD-8 alpha, CD28,4-1BB, OX40, CD3-zeta, T cell receptor α or β chain, a CD3 zeta chain,CD28, CD3ε, CD45, CD4, CD5, CD8, CD8a, CD9, CD16, CD22, CD33, CD37,CD64, CD80, CD86, CD134, CD137, ICOS, CD154, functional derivativesthereof, and combinations thereof.

In one embodiment the hinge region includes the CD8 a hinge region.

In some embodiments, the hinge region includes one selected from, butnot limited to, immunoglobulin (e.g. IgG1, IgG2, IgG3, IgG4, and IgD).

The transmembrane domain includes a hydrophobic polypeptide that spansthe cellular membrane. In particular, the transmembrane domain spansfrom one side of a cell membrane (extracellular) through to the otherside of the cell membrane (intracellular or cytoplasmic).

The transmembrane domain may be in the form of an alpha helix or a betabarrel, or combinations thereof. The transmembrane domain may include apolytopic protein, which has many transmembrane segments, eachalpha-helical, beta sheets, or combinations thereof.

In one embodiment, the transmembrane domain that is naturally associatedwith one of the domains in the CAR is used. In another embodiment, thetransmembrane domain is selected or modified by amino acid substitutionto avoid binding of such domains to the transmembrane domains of thesame or different surface membrane proteins to minimize interactionswith other members of the receptor complex.

For example, a transmembrane domain includes a transmembrane domain of aT-cell receptor a or 3 chain, a CD3 zeta chain, CD28, CD3ε, CD45, CD4,CD5, CD7, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD68,CD134, CD137, ICOS, CD41, CD154, functional derivatives thereof, andcombinations thereof.

In one embodiment, the transmembrane domain is artificially designed sothat more than 25%, more than 50% or more than 75% of the amino acidresidues of the domain are hydrophobic residues such as leucine andvaline. In one embodiment, a triplet of phenylalanine, tryptophan andvaline is found at each end of the synthetic transmembrane domain.

In one embodiment, the transmembrane domain is the CD8 transmembranedomain. In another embodiment, the transmembrane domain is the CD28transmembrane domain. Such transmembrane domains are known in the art.

The signaling domain and co-stimulatory domain include polypeptides thatprovide activation of an immune cell to stimulate or activate at leastsome aspect of the immune cell signaling pathway.

In an embodiment, the signaling domain includes the polypeptide of afunctional signaling domain of CD3 zeta, common FcR gamma (FCER1G), Fcgamma Rlla, FcR beta (Fc Epsilon Rib), CD3 gamma, CD3 delta, CD3epsilon, CD79a, CD79b, DNAX-activating protein 10 (DAP10),DNAX-activating protein 12 (DAP12), active fragments thereof, functionalderivatives thereof, and combinations thereof. Such signaling domainsare known in the art.

In an embodiment, the CAR polypeptide further includes one or moreco-stimulatory domains. In an embodiment, the co-stimulatory domain is afunctional signaling domain from a protein including OX40; CD27; CD28;CD30; CD40; PD-1; CD2; CD7; CD258; Natural killer Group 2 member C(NKG2C); Natural killer Group 2 member D (NKG2D), B7-H3; a ligand thatbinds to at least one of CD83, ICAM-1, LFA-1 (CD11a/CD18), ICOS, and4-1BB (CD137); CDS; ICAM-1; LFA-1 (CD1a/CD18); CD40; CD27; CD7; B7-H3;NKG2C; PD-1; ICOS; active fragments thereof; functional derivativesthereof; and combinations thereof.

As used herein, the at least one co-stimulatory domain and signalingdomain may be collectively referred to as the intracellular domain. Asused herein, the hinge region and the antigen recognition domain may becollectively referred to as the extracellular domain.

The present disclosure further provides a polynucleotide encoding thechimeric antigen receptor polypeptide described above.

The term “polynucleotide” as used herein is defined as a chain ofnucleotides. Polynucleotide includes DNA and RNA. Furthermore, nucleicacids are polymers of nucleotides. Thus, nucleic acids andpolynucleotides as used herein are interchangeable. One skilled in theart has the general knowledge that nucleic acids are polynucleotides,which can be hydrolyzed into the monomeric “nucleotides.” The monomericnucleotides can be hydrolyzed into nucleosides. As used hereinpolynucleotides include, but are not limited to, all nucleic acidsequences which are obtained by any means available in the art,including, without limitation, recombinant means, i.e., the cloning ofnucleic acid sequences from a recombinant library or a cell genome,using ordinary cloning technology and polymerase chain reaction (PCR),and the like, and by synthetic means.

The polynucleotide encoding the CAR is easily prepared from an aminoacid sequence of the specified CAR by any conventional method. A basesequence encoding an amino acid sequence can be obtained from theaforementioned NCBI RefSeq IDs or accession numbers of GenBenk for anamino acid sequence of each domain, and the nucleic acid of the presentdisclosure can be prepared using a standard molecular biological and/orchemical procedure. For example, based on the base sequence, apolynucleotide can be synthesized, and the polynucleotide of the presentdisclosure can be prepared by combining DNA fragments which are obtainedfrom a cDNA library using a polymerase chain reaction (PCR).

In one embodiment, the polynucleotide disclosed herein is part of agene, or an expression or cloning cassette.

The polynucleotide described above can be cloned into a vector. A“vector” is a composition of matter which includes an isolatedpolynucleotide and which can be used to deliver the isolatedpolynucleotide to the interior of a cell. Numerous vectors are known inthe art including, but not limited to, linear polynucleotides,polynucleotides associated with ionic or amphiphilic compounds,plasmids, phagemid, cosmid, and viruses. Viruses include phages, phagederivatives. Thus, the term “vector” includes an autonomouslyreplicating plasmid or a virus. The term should also be construed toinclude non-plasmid and non-viral compounds which facilitate transfer ofnucleic acid into cells, such as, for example, polylysine compounds,liposomes, and the like. Examples of viral vectors include, but are notlimited to, adenoviral vectors, adeno-associated virus vectors,retroviral vectors, lentiviral vectors, and the like. In one embodiment,vectors include cloning vectors, expression vectors, replicationvectors, probe generation vectors, integration vectors, and sequencingvectors.

In an embodiment, the vector is a viral vector. In an embodiment, theviral vector is a retroviral vector or a lentiviral vector. In anembodiment, the engineered cell is virally transduced to express thepolynucleotide sequence.

A number of viral based systems have been developed for gene transferinto mammalian cells. For example, retroviruses provide a convenientplatform for gene delivery systems. A selected gene can be inserted intoa vector and packaged in retroviral particles using techniques known inthe art. The recombinant virus can then be isolated and delivered tocells of the patient either in vivo or ex vivo. A number of retroviralsystems are known in the art. In some embodiments, adenovirus vectorsare used. A number of adenovirus vectors are known in the art. In oneembodiment, lentivirus vectors are used.

Viral vector technology is well known in the art and is described, forexample, in Sambrook et al, (2001, Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory, New York), and in other virologyand molecular biology manuals. Viruses, which are useful as vectorsinclude, but are not limited to, retroviruses, adenoviruses,adeno-associated viruses, herpes viruses, and lentiviruses. In general,a suitable vector contains an origin of replication functional in atleast one organism, a promoter sequence, convenient and uniquerestriction endonuclease sites, and one or more selectable markers,(e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193).

Lentiviral vectors have been well known for their capability oftransferring genes into human T cells with high efficiency butexpression of the vector-encoded genes is dependent on the internalpromoter that drives their expression. A strong promoter is particularlyimportant for the third or fourth generation of CARs that bearadditional co-stimulatory domains or genes encoding proliferativecytokines as increased CAR body size does not guarantee equal levels ofexpression. There are a wide range of promoters with different strengthand cell-type specificity. Gene therapies using CAR T cells rely on theability of T cells to express adequate CAR body and maintain expressionover a long period of time. The EF-1α promoter has been commonlyselected for the CAR expression.

The present disclosure provides an expression vector containing a strongpromoter for high level gene expression in T cells or NK cells. Infurther embodiment, the inventor discloses a strong promoter useful forhigh level expression of CARs in T cells or NK cells. In particularembodiments, a strong promoter relates to the SFFV promoter, which isselectively introduced in an expression vector to obtain high levels ofexpression and maintain expression over a long period of time in T cellsor NK cells. Expressed genes prefer CARs, T cell co-stimulatory factorsand cytokines used for immunotherapy.

One example of a suitable promoter is the immediate earlycytomegalovirus (CMV) promoter sequence. This promoter sequence is astrong constitutive promoter sequence capable of driving high levels ofexpression of any polynucleotide sequence operatively linked thereto.Another example of a suitable promoter is Elongation Growth Factor-1 a(EF-1 a). However, other constitutive promoter sequences may also beused, including, but not limited to the simian virus 40 (SV40) earlypromoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus(HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avianleukemia virus promoter, an Epstein-Barr virus immediate early promoter,a Rous sarcoma virus promoter, as well as human gene promoters such as,but not limited to, the actin promoter, the myosin promoter, thehemoglobin promoter, and the creatine kinase promoter. Further, thedisclosure should not be limited to the use of constitutive promoters,inducible promoters are also contemplated as part of the disclosure. Theuse of an inducible promoter provides a molecular switch capable ofturning on expression of the polynucleotide sequence, which isoperatively linked when such expression is desired, or turning off theexpression when expression is not desired. Examples of induciblepromoters include, but are not limited to a metalothionine promoter, aglucocorticoid promoter, a progesterone promoter, and a tetracyclinepromoter.

Expression of chimeric antigen receptor polynucleotide may be achievedusing, for example, expression vectors including, but not limited to, atleast one of a SFFV (spleen-focus forming virus) (for example, SEQ IDNO. 23) or human elongation factor 11a (EF) promoter, CAG (chickenbeta-actin promoter with CMV enhancer) promoter human elongation factor1a (EF) promoter. Examples of less-strong/lower-expressing promotersutilized may include, but is not limited to, the simian virus 40 (SV40)early promoter, cytomegalovirus (CMV) immediate-early promoter,Ubiquitin C (UBC) promoter, and the phosphoglycerate kinase 1 (PGK)promoter, or a part thereof. Inducible expression of chimeric antigenreceptor may be achieved using, for example, a tetracycline responsivepromoter, including, but not limited to, TRE3GV (Tet-response element,including all generations and preferably, the 3rd generation), induciblepromoter (Clontech Laboratories, Mountain View, CA) or a part or acombination thereof.

In a preferred embodiment, the promoter is an SFFV promoter or aderivative thereof. It has been unexpectedly discovered that SFFVpromoter provides stronger expression and greater persistence in thetransduced cells in accordance with the present disclosure.

“Expression vector” refers to a vector including a recombinantpolynucleotide comprising expression control sequences operativelylinked to a nucleotide sequence to be expressed. An expression vectorincludes sufficient cis-acting elements for expression; other elementsfor expression can be supplied by the host cell or in an in vitroexpression system. Expression vectors include all those known in theart, such as cosmids, plasmids (e.g., naked or contained in liposomes)and viruses (e.g., lentiviruses, retroviruses, adenoviruses, andadeno-associated viruses) that incorporate the recombinantpolynucleotide. The expression vector may be a bicistronic ormulticistronic expression vector. Bicistronic or multicistronicexpression vectors may include (1) multiple promoters fused to each ofthe open reading frames; (2) insertion of splicing signals betweengenes; fusion of genes whose expressions are driven by a singlepromoter; (3) insertion of proteolytic cleavage sites between genes(self-cleavage peptide); and (iv) insertion of internal ribosomal entrysites (IRESs) between genes.

In one embodiment, the disclosure provides an engineered cell having atleast one chimeric antigen receptor polypeptide or polynucleotide.

An “engineered cell” means any cell of any organism that is modified,transformed, or manipulated by addition or modification of a gene, a DNAor RNA sequence, or protein or polypeptide. Isolated cells, host cells,and genetically engineered cells of the present disclosure includeisolated immune cells, such as NK cells and T cells that contain the DNAor RNA sequences encoding a chimeric antigen receptor or chimericantigen receptor complex and express the chimeric receptor on the cellsurface. Isolated host cells and engineered cells may be used, forexample, for enhancing an NK cell activity or a T lymphocyte activity,treatment of cancer, and treatment of infectious diseases.

In an embodiment, the engineered cell includes immunoregulatory cells.Immunoregulatory cells include T-cells, such as CD4 T-cells (HelperT-cells), CD8 T-cells (Cytotoxic T-cells, CTLs), and memory T cells ormemory stem cell T cells. In another embodiment, T-cells include NaturalKiller T-cells (NK T-cells).

In an embodiment, the engineered cell includes Natural Killer cells.Natural killer cells are well known in the art. In one embodiment,natural killer cells include cell lines, such as NK-92 cells. Furtherexamples of NK cell lines include NKG, YT, NK-YS, HANK-1, YTS cells, andNKL cells.

NK cells mediate anti-tumor effects without the risk of GvHD and areshort-lived relative to T-cells. Accordingly, NK cells would beexhausted shortly after destroying cancer cells, decreasing the need foran inducible suicide gene on CAR constructs that would ablate themodified cells.

In accordance with the present disclosure, it was surprisingly foundthat NK cells provide a readily available cell to be engineered tocontain and express the chimeric antigen receptor polypeptides disclosedherein.

Allogeneic or autologous NK cells induce a rapid immune response butdisappear relatively rapidly from the circulation due to their limitedlifespan. Thus, applicants surprisingly discovered that there is reducedconcern of persisting side effects using CAR cell based therapy.

According to one aspect of the present disclosure, NK cells can beexpanded and transfected with CAR polynucleotides in accordance to thepresent disclosure. NK cells can be derived from cord blood, peripheralblood, iPS cells and embryonic stem cells. According to one aspect ofthe present disclosure, NK-92 cells may be expanded and transfected withCAR. NK-92 is a continuously growing cell line that has features andcharacteristics of natural killer (NK) cells (Arai, Meagher et al.2008). NK-92 cell line is IL-2 dependent and has been proven to be safe(Arai, Meagher et al. 2008) and feasible. CAR expressing NK-92 cells canbe expanded in the serum free-medium with or without co-culturing withfeeder cells. A pure population of NK-92 carrying the CAR of interestmay be obtained by sorting.

In one embodiment, engineered cells include allogeneic T cells obtainedfrom donors that are modified to inactivate components of TCR (T cellreceptor) involved in MHC recognition. As a result, TCR deficient Tcells would not cause graft versus host disease (GVHD).

In some embodiments, the engineered cell may be modified to preventexpression of cell surface antigens. For example, an engineered cell maybe genetically modified to delete the native CD45 gene to preventexpression and cell surface display thereof.

In some embodiments, the engineered cell includes an inducible suicidegene (“safety switch”) or a combination of safety switches, which may beassembled on a vector, such as, without limiting, a retroviral vector,lentiviral vector, adenoviral vector or plasmid. Introduction of a“safety switch” greatly increases safety profile and limits on-target oroff-tumor toxicities of the compound CARs. The “safety switch” may be aninducible suicide gene, such as, without limiting, caspase 9 gene,thymidine kinase, cytosine deaminase (CD) or cytochrome P450. Othersafety switches for elimination of unwanted modified T cells involveexpression of CD20 or CD52 or CD19 or truncated epidermal growth factorreceptor in T cells. All possible safety switches have been contemplatedand are embodied in the present disclosure.

In some embodiments, the suicide gene is integrated into the engineeredcell genome.

In one embodiment, the present disclosure provides an engineered cellhaving a CD45 chimeric antigen receptor polynucleotide. In oneembodiment, the CD45 CAR polypeptide includes SEQ ID NO. 13 andcorresponding polynucleotide sequence SEQ ID NO. 14. In anotherembodiment, the CD45 CAR polypeptide includes SEQ ID NO. 15, andcorresponding polynucleotide sequence SEQ ID NO. 16. In anotherembodiment, the CD45 CAR polypeptide includes SEQ ID NO. 17, andcorresponding polynucleotide sequence SEQ ID NO. 18.

In particular embodiments, the engineered cell includes CD45 CAR linkedto IL15/IL-15sushi via the P2A cleavage sequence. A polypeptideproviding this embodiment includes SEQ ID No. 43 and correspondingpolynucleotide sequence SEQ ID No. 44.

In particular embodiments, the engineered cell includes CD45 CAR linkedto 4-1BBL (CD137L) via the P2A cleavage sequence. A polypeptideproviding this embodiment includes SEQ ID No. 41 and correspondingpolynucleotide sequence SEQ ID No. 42.

Multiple CAR Units

In one embodiment, the present disclosure provides an engineered cellhaving at least two distinct or separate CAR units. The two CAR unitsmay be complete CAR units or incomplete CAR units. As used herein,“distinct CAR polypeptide” and “distinct CAR polypeptide unit” are usedinterchangeably.

The present disclosure provides chimeric antigen receptor polypeptideshaving a signal peptide, antigen recognition domain, a hinge region, atransmembrane domain, a signaling domain, and at least oneco-stimulatory domain, defining a CAR unit or a complete CAR unit. Asused herein, an incomplete CAR unit includes a polypeptide having asignal peptide, antigen recognition domain, a hinge region, atransmembrane domain, and a signaling domain or at least oneco-stimulatory domain. An incomplete CAR unit will not contain asignaling domain and at least one co-stimulatory domain, but one or theother.

In one embodiment, the present disclosure provides an engineered cellhaving a first chimeric antigen receptor polypeptide having a firstantigen recognition domain and a co-stimulatory domain (first incompleteCAR unit); and a second chimeric antigen receptor polypeptide having asecond antigen recognition domain and a signaling domain (secondincomplete CAR unit); wherein the first antigen recognition domain isdifferent than the second antigen recognition domain.

Therefore, an engineered cell having two incomplete CAR units will onlybe fully activated when both target antigens are bound to the antigenrecognition domain. This strategy provides added specificity in that theengineered cells are not fully activated until targets are bound at theantigen recognition domain of each incomplete CAR unit.

Furthermore, in embodiments wherein an engineered cell includes twoincomplete CAR units, one of the antigen recognition domains may bespecific for and bind streptavidin, biotin, HIS, MYC, HA, agarose, V5,Maltose, GST, GFP, CD52, CD20, 4-1BB, or CD28.

As used herein, compound CAR (cCAIR) or multiple CAR refers to anengineered cell having at least two complete and distinct chimericantigen receptor polypeptides. As used herein, a “distinct chimericantigen receptor polypeptide” has a unique antigen recognition domain, asignal peptide, a hinge region, a transmembrane domain, at least onecostimulatory domain, and a signaling domain. Therefore, two uniquechimeric antigen receptor polypeptides will have different antigenrecognition domains. The signal peptide, hinge region, transmembranedomain, at least one costimulatory domain, and signaling domain may bethe same or different between the two distinct chimeric antigen receptorpolypeptides. As used herein, a chimeric antigen receptor (CAR) unitrefers to a distinct chimeric antigen receptor polypeptide, or apolynucleotide encoding for the same.

As used herein, a unique antigen recognition domain is one that isspecific for or targets a single target, or a single epitope of atarget.

In some embodiments, the compound CAR targets the same antigen. Forexample, cCAR targets different epitopes or parts of a single antigen.In some embodiments, each of the CAR units present in the compound CARtargets different antigen specific to the same or different diseasecondition or side effects caused by a disease condition.

In some embodiments, the compound CAR targets two different antigens.

Creation of compound CARs bearing different CAR units can be quitechallenging: (1) CAR-CAR interactions might have a deleterious effectand an appropriate CAR design is a key to offset this effect; (2) acompound CAR in a single construct could increase the length of theexpression cassette, which may cause the reduction of the viral titerand level of protein expression; (3) an appropriate design to includevarious CAR body elements particularly to select a strategy to expressmultiple CARs in a single vector is required; (4) A strong promoter isparticularly important for a compound CAR that bears additional units ofCAR; (5) The hinge region in the CAR needs to be designed so thatinteraction of the hinge region between each CAR unit is avoidedpreferably; (6) two or more units of CARs expressing in a cell may causetoxic effects (CAR-CAR interaction). Applicants herein provide novel andsurprising CAR compositions and methods to overcome these hurdles.

In one embodiment, the present disclosure provides an engineered cellhaving multiple CAR units. This allows a single engineered cell totarget multiple antigens. Targeting multiple surface markers or antigenssimultaneously with a multiple CAR unit prevents selection of resistantclones and reduces tumor recurrence. Multiple CAR T cellimmunotherapies, with each individual component CAR comprising variousdomains and activation sites has not yet been developed for anymalignancies.

In one aspect of the present disclosure, cCAR includes multiple CARunits. In some embodiments, cCAR includes at least two CAR units. Inanother embodiment, the cCAR includes at least three CAR units. Inanother embodiment, the cCAR includes at least four units.

In one embodiment, the present disclosure provides an engineered cellhaving at least two distinct chimeric antigen receptor polypeptides,each having a different antigen recognition domain.

In one embodiment, the engineered cell having at least two distinctchimeric antigen receptor polypeptides is a T-cell. The T-cell may beengineered so that it does not express a cell surface antigen. Forexample, a T-cell may be engineered so that it does not express a CD45cell surface antigen.

In a preferred embodiment, the engineered cell having at least twodistinct chimeric antigen receptor polypeptides is a primary NK cellisolated from the peripheral blood or cord blood and NK-92 cells, suchthat it is administered “off-the-shelf” to any mammal with a disease orcancer.

In one embodiment, the engineered cell includes (i.) a first chimericantigen receptor polypeptide comprising a first antigen recognitiondomain, a first signal peptide, a first hinge region, a firsttransmembrane domain, a first co-stimulatory domain, and a firstsignaling domain; and (ii.) a second chimeric antigen receptorpolypeptide comprising a second antigen recognition domain, a secondsignal peptide, a second hinge region, a second transmembrane domain, asecond co-stimulatory domain, and a second signaling domain. The firstantigen recognition domain is different from the second antigenrecognition domain.

In a preferred embodiment, each engineered CAR unit polynucleotide havedifferent nucleotide sequences in order to avoid homologousrecombination.

In one embodiment, the target of the first antigen recognition domain isselected from the group consisting of interleukin 6 receptor, NY-ESO-1,alpha fetoprotein (AFP), glypican-3 (GPC3), BAFF-R, BCMA, TACI, LeY,CD5, CD13, CD14, CD15 CD19, CD20, CD22, CD33, CD41, CD61, CD64, CD68,CD117, CD123, CD138, CD267, CD269, CD38, Flt3 receptor, and CS1; and thetarget of the second recognition domain is selected from the groupconsisting of interleukin 6 receptor, NY-ESO-1, alpha fetoprotein (AFP),glypican-3 (GPC3), BAFF-R, BCMA, TACI, LeY, CD5, CD13, CD14, CD15, CD19,CD20, CD22, CD33, CD41, CD61, CD64, CD68, CD117, CD123, CD138, CD267,CD269, CD38, Flt3 receptor, and CS1.

In one embodiment, the engineered cell includes a first chimeric antigenreceptor polypeptide having a CD19 antigen recognition domain and secondchimeric antigen receptor polypeptide having a CD20 recognition domain.In one embodiment, this engineered cell includes a polypeptide of SEQ IDNO. 3 and corresponding polynucleotide of SEQ ID NO. 4.

In one embodiment, the engineered cell includes a first chimeric antigenreceptor polypeptide having a CD19 antigen recognition domain and secondchimeric antigen receptor polypeptide having a CD22 antigen recognitiondomain. In one embodiment, this engineered cell includes a polypeptideof SEQ ID NO. 5 and corresponding polynucleotide of SEQ ID NO. 6.

In one embodiment, the engineered cell includes a first chimeric antigenreceptor polypeptide having a CD19 antigen recognition domain and secondchimeric antigen receptor polypeptide having a CD123 antigen recognitiondomain. In one embodiment, this engineered cell includes a polypeptideof SEQ ID NO. 7 and corresponding polynucleotide of SEQ ID NO. 8.

In one embodiment, the engineered cell includes a first chimeric antigenreceptor polypeptide having a CD33 antigen recognition domain and secondchimeric antigen receptor polypeptide having a CD123antigen recognitiondomain. In one embodiment, this engineered cell includes a polypeptideof SEQ ID NO. 9 and corresponding polynucleotide of SEQ ID NO. 10. Inanother embodiment, this engineered cell includes a polypeptide of SEQID NO. 11 and corresponding polynucleotide of SEQ ID NO. 12.

In one embodiment, the engineered cell includes a first chimeric antigenreceptor polypeptide having a BAFF-R antigen recognition domain andsecond chimeric antigen receptor polypeptide having a CS1antigenrecognition domain.

In one embodiment, the engineered cell includes a first chimeric antigenreceptor polypeptide having a CD269 antigen recognition domain andsecond chimeric antigen receptor polypeptide having a CS1 antigenrecognition domain. In one embodiment, the engineered cell includes apolypeptide including SEQ ID NO. 19 and corresponding polynucleotide SEQID NO. 20. In one embodiment, the engineered cell includes a polypeptideincluding SEQ ID NO. 21 and corresponding polynucleotide SEQ ID NO. 22.

In one embodiment, the engineered cell includes a first chimeric antigenreceptor polypeptide having a CD33 antigen recognition domain and secondchimeric antigen receptor polypeptide having a CD123 antigen recognitiondomain.

In one embodiment, each CAR unit includes the same or different hingeregion. In another embodiment, each CAR unit includes the same ordifferent transmembrane region. In another embodiment, each CAR unitincludes the same or different intracellular domain.

In one embodiment, each CAR unit includes the CD3 zeta chain signalingdomain.

In one embodiment, each distinct CAR unit includes differentco-stimulatory domains to avoid interaction. For example, the firstchimeric antigen receptor polypeptide includes a 4-BB co-stimulatorydomain; and the second chimeric antigen receptor polypeptide includes aCD28 co-stimulatory domain.

In another embodiment, the hinge region is designed to exclude aminoacids that may cause undesired intra- or intermolecular interactions.For example, the hinge region may be designed to exclude or minimizecysteine residues to prevent formation of disulfide bonds. In anotherembodiment, the hinge region may be designed to exclude or minimizehydrophobic residues to prevent unwanted hydrophobic interactions.

Compound CAR can perform killing independently or in combination.Multiple or compound CAR comprises same or different hinge region, sameor different transmembrane, same or different co-stimulatory and same ordifferent intracellular domains. Preferably, the hinge region isselected to avoid the interaction site.

The compound CAR of the present disclosure may target same or differenttumor populations in T or NK cells. The first CAR, for example, maytarget the bulky tumor population and the next or the second CAR, forexample, may eradicate cancer or leukemic stem cells, to avoid cancerrelapses.

In accordance with the present disclosure it was surprisingly found thatthe compound CAR in a T or NK cells targeting different or same tumorpopulations combat tumor factors causing cancer cells resistant to theCAR killing activity, thereby producing down regulation of the targetantigen from the cancer cell surface. It was also surprisingly foundthat this enables the cancer cell to “hide” from the CAR therapyreferred to as “antigen escape” and tumor heterogeneity, by whichdifferent tumor cells can exhibit distinct surface antigen expressionprofiles.

Engineered Cell Having CAR Polypeptide and Enhancer

In another embodiment, the present disclosure provides an engineeredcell having at least one chimeric antigen receptor polypeptide and anenhancer.

In one embodiment, the present disclosure provides an engineered cellhaving at least two distinct chimeric antigen receptor polypeptides andan enhancer.

As used herein, an enhancer includes a biological molecule that promotesor enhances the activity of the engineered cell having the chimericantigen receptor polypeptide. Enhancers include cytokines. In anotherembodiment, enhancers include IL-2, IL-7, IL-12, IL-15, IL-18, IL-21,PD-1, PD-L1, CSF1R, CTAL-4, TIM-3, and TGFR beta, receptors for thesame, and functional fragments thereof.

Enhancers may be expressed by the engineered cell described herein anddisplayed on the surface of the engineered cell or the enhancer may besecreted into the surrounding extracellular space by the engineeredcell. Methods of surface display and secretion are well known in theart. For example, the enhancer may be a fusion protein with a peptidethat provides surface display or secretion into the extracellular space.

The effect of the enhancer may be complemented by additional factorssuch as enhancer receptors and functional fragments thereof. Theadditional factors may be co-expressed with the enhancer as a fusionprotein, or expressed as a separate polypeptide and secreted into theextracellular space.

Enhancers can be cytokines secreted from engineered CAR cells and aredesigned to co-express with the CAR polypeptide. A massive releaseoccurs upon CAR engagement of cognate antigen. Inflammatory cellssurrounding tumor cells have a significant correlation with cancer cellprogression and metastasis. Inflammatory cells could include T cells andinnate immune response cells, such as NK cells, macrophages, anddendritic cells and their proliferation and anti-tumor activity areregulated by cytokines. CAR cells such as CAR T or NK cells bind totargeted cancer cells and trigger massive secretion of enhancers fromthe expansion of CAR T/NK cells. The secreted enhancers efficientlypromote survival, differentiation and activation of immune responsecells against cancer cells. The co-expression of an enhancer(s) with CARcan supplement the defect that CAR T or NK cells are unable to eliminatenon-targeting cancer cells (FIG. 78 ).

CAR cells can be a carrier of cytokines, and cytokines can be deliveredto targeted cancer sites by CAR cells to reduce systemic toxicity withhigh-dose exogenous cytokines (FIG. 78 ).

To improve sustained survival or long-lived persistence of CAR cells, amembrane bound enhancer (s) can be co-expressed with CAR to improve CARpersistency.

In one embodiment, the enhancer is IL-15. In this instance, theadditional factor described above is the IL-15 receptor, and functionalfragments thereof. Functional fragments include the IL-15 receptor,IL-15RA, and the sushi domain of IL-15RA (IL-15sushi). Soluble IL-15RAor IL15sushi profoundly potentiates IL-15 functional activity byprevention of IL-15 degradation. Soluble IL-15/IL-15RA orIL-15/IL-15sushi complexes are stable and much more stimulatory thanIL-15 alone in vivo.

In one embodiment, IL-15 is co-expressed as a fusion protein with atleast one of IL-15 receptor, IL-15RA, and the sushi domain of IL-15RA(IL-15sushi). In one embodiment, the IL-15 receptor, IL-15RA, or thesushi domain of IL-15RA (IL-15sushi) is at the N-terminus of IL-15. Inanother embodiment, the IL-15 receptor, IL-15RA, or the sushi domain ofIL-15RA (IL-15sushi) is at the C-terminus of IL-15. As used herein,IL-15/IL-15 sushi denotes that IL-15 sushi is at the C-terminus of IL-15in a fusion protein; and IL-15sushi/il-15 denotes that IL-15 sushi is atthe N-terminus of IL-15 in a fusion protein.

In some embodiments, IL-15 and the IL-15 receptor or functionalfragments thereof polypeptide is on a single polypeptide molecule and isseparated by a peptide linker, the peptide linker may be 1-25 amino acidresidues in length, 25-100 amino acid residues in length, or 50-200amino acid residues in length. This linker may include a high efficiencycleavage site described herein.

An example of a suitable sushi domain includes a CAR construct, SEQ IDNO. 1. In accordance with the present disclosure, any chimeric antigenreceptor polypeptide disclosed herein may be co-expressed with the HumanInterleukin 15 with human interleukin 2 signal peptide SEQ ID NO. 2.

Interleukin (IL)-15 and its specific receptor chain, IL-15Rα (IL-15-RA)play a key functional role in various effector cells, including NK andCD8 T cells. CD8+ T cells can be modified to express autocrine growthfactors including, but not limited to, IL-2, I1-7, IL21 or IL-15, tosustain survival following transfer in vivo. Without wishing to be boundby theory, it is believed that IL-15 overcomes the CD4 deficiency toinduce primary and recall memory CD8T cells. Overexpression of IL-15-RAor an IL-15 IL-RA fusion on CD8 T cells significantly enhances itssurvival and proliferation in-vitro and in-vivo. In some embodiments,CD4CAR or any CAR is co-expressed with at least one of IL-15, IL15RA andIL-15/IL-15R or IL15-RA/IL-15, or a part or a combination thereof, toenhance survival or proliferation of CAR T or NK, and to improveexpansion of memory CAR CD8+ T cells.

The present disclosure provides an engineered cell having a CARpolypeptide as described herein and at least one of IL-15, IL-15RA,IL-15sushi, IL-15/IL-15RA, IL15-RA/IL-15, IL-15/IL-15sushi,IL15sushi/IL-15, fragment thereof, a combination thereof, to enhancesurvival or persistence or proliferation of CAR T or NK for treatingcancer in a patient.

In another embodiment, the present disclosure provides an engineeredcell having at least one of recombinant IL-15, IL-15RA, IL-15sushi,IL-15/IL-15RA, IL15-RA/IL-15, IL-15/IL-15sushi, IL15sushi/IL-15,functional fragment thereof, and combination thereof; and at least onedistinct CAR polypeptide wherein the antigen recognition domain includesNY-ESO-1, alpha fetoprotein (AFP), glypican-3 (GPC3), BCMA, BAFF-R,BCMA, TACI, LeY, CD5, CD7, CD2, CD3, CD4, CD45, CD13, CD14, CD15, CD19,CD20, CD22, CD33, CD41, CD61, CD64, CD68, CD117, CD123, CD138, CD267,CD269, CD38, Flt3 receptor, ROR1, PSMA, MAGE A3, Glycolipid, F77, GD-2,WT1, CEA, HER-2/neu, MAGE-3, MAGE-4, MAGE-5, MAGE-6, CA 19-9, CA 72-4,NY-ESO, FAP, ErbB, c-Met, MART-1, CD30, EGFRvIII, immunoglobin kappa andlambda, CD38 and CS1. The target antigens can also include viral orfungal antigens, such as E6 and E7 from the human papillomavirus (HPV)or EBV (Epstein Barr virus) antigens. In further embodiment, the antigenrecognition polypeptides (scFv) and corresponding polynucleotides forCD2, CD3, CD5, CD7, and CD52 are described in more detail publicationsin PCT Application NO. PCT/US2016/39306, the contents of which areincorporated herein by reference.

Without wishing to be bound by theory, it is believed thatIL-15/IL-15sushi and other types of IL-15 or IL-15RA proteins or proteinfragments thereof provide synergistic efficacy of a CAR polypeptide whencombined with checkpoint inhibitors or modulators (e.g. anti-PD-1).

In one embodiment, the disclosure provides a CD4 CAR engineered cellthat includes IL-15/IL-15sushi (SEQ ID NO. 1), and correspondingpolynucleotide (SEQ ID NO. 2). In one embodiment, the present disclosureprovides a method of providing long-term durable remission in cancerpatients by administering a CD4 CAR engineered cell that includesIL-15/IL-15sushi to a patient in need thereof. Without wishing to bebound by theory, it is believed that co-expression of IL-15/IL-15sushiwith a CD4 CAR polypeptide provides long-term durable remission inpatients by increasing the sensitivity of CAR recognition of targetcancer cells or recruiting innate immune cells to cancer cells.

In one embodiment, the present disclosure provides engineered cellhaving a CD45 chimeric antigen receptor polypeptide and IL-15/IL-15sushi(SEQ ID NO. 44), and corresponding nucleotides (SEQ ID NO. 43).

In one embodiment, the present disclosure provides a method of providinglong-term durable remission in cancer patients by administering a CD45CAR engineered cell that includes IL-15/IL-15sushi to a patient in needthereof. Without wishing to be bound by theory, it is believed thatco-expression of IL-15/IL-15sushi with a CD45 CAR polypeptide provideslong-term durable remission in patients by increasing the sensitivity ofCAR recognition of target cancer cells or recruiting innate immune cellsto cancer cells.

In one embodiment, the engineered cell includes a CD45 chimeric antigenreceptor polypeptide and 4-1BBL (SEQ ID NO. 74), and correspondingnucleotides (SEQ ID NO. 73).

In one embodiment, the present disclosure provides a method of providinglong-term durable remission in patients suffering from cancer byadministering a CD45 CAR engineered cell that co-expresses 4-1BBL to apatient in need thereof. Without wishing to be bound by theory, it isbelieved that co-expression of 4-1BBL with a CD45 CAR provides long-termdurable remission in patients by increasing the persistence of CARengineered cells.

In one embodiment, the engineered cell includes a CD19 chimeric antigenreceptor polypeptide and IL-15/IL-15sushi (SEQ ID NO. 59), andcorresponding polynucleotide (SEQ ID NO. 60). In one embodiment, thepresent disclosure provides a method of providing long-term durableremission in cancer patients by administering a CD19 CAR engineered cellthat includes IL-15/IL-15sushi to a patient in need thereof. Withoutwishing to be bound by theory, it is believed that co-expression ofIL-15/IL-15sushi with a CD19 CAR provides long-term durable remission inpatients by increasing the sensitivity of CAR recognition of targetcancer cells or recruiting innate immune cells to cancer cells.

In one embodiment, the engineered cell includes a CD20 chimeric antigenreceptor polypeptide and IL-15/IL-15sushi (SEQ ID NO.58), andcorresponding polynucleotide (SEQ ID NO. 57). In one embodiment, thepresent disclosure provides a method of providing long-term durableremission in cancer patients by administering a CD20 CAR engineered cellthat includes IL-15/IL-15sushi to a patient in need thereof. Withoutwishing to be bound by theory, it is believed that co-expression ofIL-15/IL-15sushi with a CD20 CAR provides long-term durable remission inpatients by increasing the sensitivity of CAR recognition of targetcancer cells or recruiting innate immune cells to cancer cells.

In one embodiment, the engineered cell includes a CD22 chimeric antigenreceptor polypeptide and IL-15/IL-15sushi (SEQ ID NO.62), andcorresponding polynucleotide (SEQ ID NO. 61). In one embodiment, thepresent disclosure provides a method of providing long-term durableremission in cancer patients by administering a CD22 CAR engineered cellthat includes IL-15/IL-15sushi to a patient in need thereof. Withoutwishing to be bound by theory, it is believed that co-expression ofIL-15/IL-15sushi with a CD22 CAR provides long-term durable remission inpatients by increasing the sensitivity of CAR recognition of targetcancer cells or recruiting innate immune cells to cancer cells.

In one embodiment, the engineered cell includes a CD269 chimeric antigenreceptor polypeptide and IL-15/IL-15sushi (SEQ ID NO. 44), andcorresponding polynucleotide (SEQ ID NO. 45). In one embodiment, thepresent disclosure provides a method of providing long-term durableremission in cancer patients by administering a CD269 CAR engineeredcell that includes IL-15/IL-15sushi to a patient in need thereof.Without wishing to be bound by theory, it is believed that co-expressionof IL-15/IL-15sushi with a CD269 CAR provides long-term durableremission in patients by increasing the sensitivity of CAR recognitionof target cancer cells or recruiting innate immune cells to cancer cellsas plasma cells or myeloma cells are usually dim CD269 (BCMA) positive.

In one embodiment, the engineered cell includes a CAR, CD4 polypeptideof SEQ ID NO. 90, and corresponding polynucleotide of SEQ ID NO. 89.

In one embodiment, the engineered cell includes a CD4 chimeric antigenreceptor polypeptide and IL-15/IL-15sushi (SEQ ID NO. 96), andcorresponding polynucleotide (SEQ ID NO. 95). In one embodiment, thepresent disclosure provides a method of providing long-term durableremission in cancer patients by administering a CD4 CAR engineered cellthat includes IL-15/IL-15sushi to a patient in need thereof. Withoutwishing to be bound by theory, it is believed that co-expression ofIL-15/IL-15sushi with a CD4 CAR provides long-term durable remission inpatients by increasing the sensitivity of CAR recognition of targetcancer cells or recruiting innate immune cells to cancer cells.

In one embodiment, the engineered cell includes a CD4 chimeric antigenreceptor polypeptide and IL-15/IL-15RA (membrane bound) (SEQ ID NO. 98),and corresponding polynucleotide (SEQ ID NO. 97). In one embodiment, thepresent disclosure provides a method of providing long-term durableremission in cancer patients by administering a CD4 CAR engineered cellthat includes IL-15/IL-15RA to a patient in need thereof. Withoutwishing to be bound by theory, it is believed that co-expression ofIL-15/IL-15RA (membrane bound) with a CD4 CAR provides long-term durableremission in patients by increasing the persistence of CAR engineeredcells.

In one embodiment, the engineered cell includes a compound CAR,CD33CD123 polypeptide and IL-15/IL-15sushi (SEQ ID NO.40), andcorresponding polynucleotide (SEQ ID NO. 39). In one embodiment, thepresent disclosure provides a method of providing long-term durableremission in cancer patients by administering a CD33CD123 compound CARengineered cell that includes IL-15/IL-15sushi to a patient in needthereof. Without wishing to be bound by theory, it is believed thatco-expression of IL-15/IL-15sushi with a CD33CD123 CAR provideslong-term durable remission in patients by increasing the sensitivity ofCAR recognition of target cancer cells or recruiting innate immune cellsto cancer cells.

In one embodiment, the engineered cell includes a compound CAR,CD33CD123 polypeptide and 4-1BBL (SEQ ID NO.38), and correspondingpolynucleotide (SEQ ID NO. 37).

In one embodiment, the present disclosure provides a method of providinglong-term durable remission in cancer patients by administering aCD33CD123 compound CAR engineered cell that co-expresses 4-1BBL to apatient in need thereof. Without wishing to be bound by theory, it isbelieved that co-expression of 4-1BBL with a CD33CD123 cCAR provideslong-term durable remission in patients by increasing the persistency ofcCAR engineered cells.

In one embodiment, the engineered cell includes a BAFF CAR polypeptidewith a CD45 leader sequence (SEQ ID NO. 78) and correspondingpolynucleotide sequence (SEQ ID NO. 77).

In one embodiment, the engineered cell includes BAFF CAR polypeptidewith a CD8a leader sequence (includes SEQ ID NO. 80) and correspondingpolynucleotide sequence (SEQ ID NO. 79).

In one embodiment, the engineered cell includes a BAFF CAR polypeptideand IL-15/IL-15sushi (SEQ ID NO. 84), and corresponding polynucleotide(SEQ ID NO. 83).

In one embodiment, the present disclosure provides a method of providinglong-term durable remission in cancer patients by administering a BAFFCAR engineered cell that includes IL-15/IL-15sushi to a patient in needthereof. Without wishing to be bound by theory, it is believed thatco-expression of IL-15/IL-15sushi with a BAFF CAR provides long-termdurable remission in patients by increasing the sensitivity of CARrecognition of target cancer cells or recruiting innate immune cells tocancer cells as BAFF receptor, CD269 (BCMA) is weakly expressed inplasma cells and myeloma cells.

In one embodiment, the engineered cell includes a BAFF CAR polypeptideand 4-1BBL (SEQ ID NO.82), and corresponding polynucleotide (SEQ ID NO.81). In one embodiment, the present disclosure provides a method ofproviding long-term durable remission in cancer patients byadministering a BAFF CAR engineered cell co-expresses 4-1BBL to apatient in need thereof. Without wishing to be bound by theory, it isbelieved that co-expression of 4-1BBL with a BAFF CAR can providelong-term durable remission in patients by increasing the persistence ofCAR engineered cells.

In one embodiment, the engineered cell includes a compound CAR, BAFFCD19b polypeptide of SEQ ID NO. 86 and corresponding polynucleotide ofSEQ ID NO. 85.

In one embodiment, the present disclosure provides a method of treatingan autoimmune disorder in a patients by administering a BAFF CD19bcompound CAR engineered cell to a patient in need thereof. Withoutwishing to be bound by theory, it is believed that the BAFF CD19bcompound CAR engineered cells provide a better therapeutic outcome fordepletion of B-cells and plasma cells associated with autoimmunedisorders.

In one embodiment, the engineered cell includes a APRIL CD19b compoundCAR polypeptide of SEQ ID NO. 88 and corresponding polynucleotide of SEQID NO. 77.

In one embodiment, the present disclosure provides a method of depletingB-cells and plasma cells in a patient in need thereof by administering aAPRIL CD19b compound CAR engineered cell to a patient in need thereof.Without wishing to be bound by theory, it is believed that the APRILCD19b compound CAR engineered cell can provide a better therapeuticoutcome for depletion of B-cells and plasma cells associated withautoimmune disorders.

In one embodiment, the engineered cell includes a compound CAR, CD269CS1 polypeptide of SEQ ID NO. 48 and corresponding polynucleotide of SEQID NO. 47. In one embodiment, the present disclosure provides a methodof treating myeloma in a patient by administering a CD269CS1 compoundCAR engineered cell to a patient in need thereof.

Without wishing to be bound by theory, it is believed that CD269 CS1compound CAR engineered cells provide a better therapeutic outcome forpatients with myeloma, and prevent antigen escape or disease relapse.

In one embodiment, the engineered cell includes a compound CAR, CD269CD19b polypeptide of SEQ ID NO. 50 and corresponding polynucleotide ofSEQ ID NO. 49.

In one embodiment, the present disclosure provides a method of depletingB-cells and plasma cells in patients by administering a CD269 CD19bcompound CAR engineered cell to a patient in need thereof. Withoutwishing to be bound by theory, it is believed that CD269 CD19b compoundCAR engineered cells provide a better therapeutic outcomes for patientssuffering from an autoimmune disorder by depletion of B-cells and plasmacells associated with autoimmune disorders.

In one embodiment, the engineered cell includes another compound CAR,CD269 CD19 polypeptide of SEQ ID NO. 52 and corresponding polynucleotideof SEQ ID NO. 51. In one embodiment, the present disclosure provides amethod of depleting B-cells and plasma cells in patients byadministering a CD269 CD19 compound CAR engineered cell to a patient inneed thereof. Without wishing to be bound by theory, it is believed thatCD269 CD19 compound CAR engineered cells provide a better therapeuticoutcomes in patients suffering from an autoimmune disorder by depletionof B-cells and plasma cells associated with autoimmune disorders.

In one embodiment, the present disclosure provides an engineered cellhaving a CD19 chimeric antigen receptor polynucleotide. In oneembodiment, the CD19 CAR polypeptide includes SEQ ID NO. 54 andcorresponding polynucleotide sequence SEQ ID NO. 53. In anotherembodiment, the CD19 CAR polypeptide includes SEQ ID NO. 56, andcorresponding polynucleotide sequence SEQ ID NO. 55 In one embodiment,the engineered cell includes a CD30 CAR polypeptide, andIL-15/IL-15sushi polypeptide (SEQ ID NO. 100), and correspondingpolynucleotide (SEQ ID NO. 99). The targeted disease is malignantHodgkin lymphoma with cancer cells expressing CD30.

In one embodiment, the present disclosure provides a method ofre-activating T-cell and innate immune cells in the tumormicroenvironment patients by administering a CD30CAR engineered cellthat secretes IL-15/IL-15 complexes to a patient in need thereof.Without wishing to be bound by theory, it is believed that IL-15/IL-15complexes (e.g. IL-15/IL-15sushi complexes) secreted from engineeredcells can re-activate T-cell and innate immune cells in the tumormicroenvironment and then restore or augment their anti-tumor immuneresponses for Hodgkin lymphoma or anaplastic large cell lymphoma.

In one embodiment, the present disclosure provides a method of restoringor augmenting T-cell or innate immune cell activation or expansionincluding coexpression of IL-15/IL-15sushi with a CAR polypeptidedisclosed herein.

In another embodiment, the disclosure provides a chimeric antigenreceptor polypeptide having an antigen recognition domain specific for aCD30 antigen.

In one embodiment, the CD30CAR includes at least one-costimulatorydomain. In another embodiment, the CD30CAR includes at least twoco-stimulatory domains.

In some embodiments, the disclosure includes a method of co-expressingIL-15/IL-15sushi with CD30CAR. In further embodiments, massive secretionof stable, functional IL-15/IL-15sushi complexes occurs upon binding ofCAR to target cells.

In another embodiment, the present disclosure provides a method oftreating a patient suffering from Hodgkin's lymphoma or a cancerassociated with a malignant cell expressing CD30 antigen byadministering a CD30 CAR engineered cell to a patient in need thereof.An example of a malignant cells expressing CD30 includes anaplasticlarge cell lymphoma.

Malignant Hodgkin lymphoma bears CD30+ Reed-Sternberg or Reed-Sternberglike cells, which are surrounded by an overwhelming numbers of T cellsand innate immune cells. These T or innate immune cells areimmunologically tolerant as they fail to eliminate cancer cells.Therefore, one of critical aspects for treating Hodgkin lymphoma is tore-activate T-cell and innate immune cells in the tumor microenvironmentand then restore or augment their anti-tumor immune responses.

In some embodiments, the present disclosure comprises a method ofco-expression of IL-15/IL-15sushi with a CD30CAR. Engineered CD30CAR Tor NK cells bind to targeted cancer cells, trigger massive secretion ofIL-15/IL-15sushi from the expansion of CD30CAR T or NK cells, wherebysecreting IL-15/IL-15sushi efficiently restore or augment T or innateimmune cells against cancer cells to overcome immunosuppressive tumormicroenvironment.

In one embodiment, the present disclosure provides a method of providinglong-term durable remission in a cancer patient by administering a CD30CAR engineered cell that co-express IL-15/IL-15sushi to a patient inneed thereof. Without wishing to be bound by theory, it is believed thatco-expression of IL-15/IL-15sushi with a CD30CAR provides long-termdurable remission in patients by increasing the sensitivity of CARrecognition of target cancer cells or recruiting innate immune cellsagainst target cancer cells to overcome immunosuppressive tumormicroenvironment.

In some embodiments, the present disclosure provides an engineered cellthat co-expresses IL-15/IL-15sushi and a CD30CAR polypeptide. Withoutwishing to be bound by theory, it is believed that the combination ofCD30CAR engineered cell with co-expression of IL-15/IL-15sushi providessynergistic efficacy when combined with checkpoint inhibitors ormodulators (e.g. anti-PD-1).

In some embodiments, the present disclosure provides a method oftreating Hodgkin's lymphoma in a patient by administering a CD30 CARengineered cell that co-expresses IL-15/IL-15sushi to a patient in needthereof. Without wishing to be bound by theory, co-expression of CD30CARpolypeptide and IL-15/IL-15sushi provides better outcomes for treatmentof Hodgkin's lymphoma or anaplastic large cells than CD30CAR alone asCD30 is not expressed in all cancer cells.

Steps for Elimination of Tumor by CAR Co-Expressing SecretoryIL-15/IL-15Sushi (FIG. 78)

In some embodiments, the present disclosure provides a method of providelong-term durable remission in a cancer patient by administering a APRILCAR engineered cell that co-expresses IL-15/IL-15sushi to a patient inneed thereof. Without wishing to be bound by theory, it is believed thatco-expression of IL-15/IL-15sushi with a APRIL CAR polypeptide provideslong-term durable remissions in patients by increasing the sensitivityof CAR recognition of target cancer cells or recruiting innate cells tocancer cells. APRIL receptor, CD269 (BCMA) is weakly expressed in plasmacells and myeloma cells.

In particular embodiments, the present disclosure provides a method forelimination of tumor cells including contacting said tumor cell with aCAR engineered cell that co-expresses IL-2 to destroy said tumor cell.

IL-15 was originally considered as an interleukin-2 (IL-2)-like factorfor T and NK cells. Unlike IL-2, IL-15 is a survival factor for memory Tcells.

In particular embodiments, elimination of tumor can be achieved bycombination of at least one or more of the following steps:

-   -   (1) binding of an CAR engineered T cell or NK cell disclosed        herein to a portion of tumor cells by targeting CAR or NK        antigen(s);    -   (2) Triggering massive secretion of IL-15/IL-15sushi or IL-2        with a prolonged half-life from expansion of CAR T/NK cells,        which co-express this molecule;    -   (3) Recruiting and stimulating a variety of innate and adaptive        immune cells against tumor;    -   (4) Reducing tumor suppression that is present in tumor by        administration of a checkpoint blockage such as PD-L1 and CTLA-4        inhibitor.

Without wishing to be bound by theory, it is believed that thecombination of steps described above provide potent anti-tumor effectsvia a concerted innate and adaptive immune response.

The engineered cells and methods described herein are suitable for thetreatment of any cancer wherein specific monoclonal or polyclonalantibodies exist or are capable of being generated in accordance withthe current state of the art. In particular, the following cancers havebeen contemplated and are considered within the scope of the presentdisclosure, neuroblastoma, lung cancer, melanoma, ovarian cancer, renalcell carcinoma, colon cancer, brain cancer, Hodgkins lymphoma, B celllymphoma/leukemia and T cell lymphoma/leukemia. All of which have cellsurface antigens that may be targeted by the chimeric antigen receptorpolypeptides and methods disclosed herein.

Many tumors escape the specific CAR T/NK killing due to the loss oftargeted antigen(s) or CAR T or NK exhaustion. The present disclosureprovides a method to overcome this escape. Without wishing to be boundby theory, the present disclosure provides a method of preventing tumorescape by administering a CAR engineered cell having an enhancer orcytokine as disclosed herein, in particular IL-15 or IL-2 to a tumorsite by CAR engineered cell. It is believed that this directlystimulates innate and adaptive immune responses. Furthermore, it isbelieved that IL-15 and/or IL-2 secretion from CAR engineered cellspromote the expansion of infused CAR T cells or CAR NK cells andinfiltration of immune cells to the tumor site, which is believed toresult in tumor destruction.

In embodiments, half-life extension and prolonged therapeutic activitycan be established in the presence of the Fc domain, such IL-15Fc orIL-2Fc. For IL-15 cytokine, IL-15/IL-15sushi or IL-15/IL-15sushi Fc ispreferred. Fc domain is referred to as the IgG Fc-domain fused tovarious effector molecules (so-called Fc-fusion proteins).

Single antigen-directed CAR immunotherapy, such as, but not limited to,CD19, CD20, CD22, CD2, CD3, CD4, CD5, CD7, CD33, CD30, CD123, CD45,BCMA, CS1, BAFF, TACI, and APRIL CAR, bears a risk of remission inpatients due to the complete loss of target antigen or changes of targetantigen expression. On this basis, the present disclosure provides amethod of providing long-term durable remission in patients byadministering an engineered cell having a CAR polypeptide disclosedherein and co-expression of IL-15/IL-15sushi to increase the sensitivityof CAR recognition of target cancer cells or recruiting innate immunecells to cancer cells.

The large volume of some solid tumors or lymphoma can be difficult forCAR T cells to eradicate the whole tumor. In addition, theimmunosuppressive microenvironment needs to be overcome as CAR T cellsmay end up simply being inactivated or suppressed when contacting tumor.

In some embodiments, the present disclosure provides a method ofco-expressing secretory IL-15/IL-15sushi and a chimeric antigen receptorpolypeptide in an engineered cell.

In some embodiments, the present disclosure provides a method ofincreasing CAR engineered cell in vivo half life by co expressingsecretory IL-15/1l-15sushi in said engineered cell. Without wishing tobe bound by theory, it is believed that the secreted complexes ofIL-15/IL-15sushi are functionally stable and efficiently promotesurvival of the CAR containing engineered cell.

In some embodiments, the present disclosure provides a method ofdelivering IL-15/IL-15sushi to targeted cancer sites using CAR as acarrier to promote the proliferation of innate immune response cellsagainst cancer cells, prevent the tumor microenvironment suppression.and reduce systemic toxicity with high-dose exogenous cytokines.

In some embodiments, the present disclosure provides a method ofdelivering IL-15/IL-15sushi to targeted cancer sites using CAR as acarrier to recruit other effector immune cells to the site and help themto kill cancer cells.

In some embodiments, the present disclosure provides a method ofdelivering IL-15/IL-15sushi to targeted cancer sites using CAR as acarrier to activate bystander immunity to eradicate cancer cells thatlose the antigen for CAR T/NK cells.

Methods of Generating Engineered Cells

Any of the polynucleotides disclosed herein may be introduced into anengineered cell by any method known in the art.

In one embodiment, CAR polynucleotides are delivered to the engineeredcell by any viral vector as disclosed herein.

In one embodiment, to achieve enhanced safety profile or therapeuticindex, the any of the engineered cells disclosed herein be constructedas a transient RNA-modified “biodegradable” version or derivatives, or acombination thereof. The RNA-modified CARs of the present disclosure maybe electroporated into T cells or NK cells. The expression of thecompound CAR may be gradually diminished over few days.

In some embodiments of the present disclosure, any of the engineeredcells disclosed herein may be constructed in a transponson system (alsocalled a “Sleeping Beauty”), which integrates the CAR DNA into the hostgenome without a viral vector.

Methods of Generating an Engineered Cell Having Multiple CAR Units

In another embodiment, the present disclosure provides a method makingan engineered cell having at least two CAR units.

In some embodiments, multiple units of CAR are expressed in a T or NKcell using bicistronic or multicistronic expression vectors. There areseveral strategies which can be employed to construct bicistronic ormulticistronic vectors including, but not limited to, (1) multiplepromoters fused to the CARs' open reading frames; (2) insertion ofsplicing signals between units of CAR; fusion of CARs whose expressionsare driven by a single promoter; (3) insertion of proteolytic cleavagesites between units of CAR (self-cleavage peptide); and (iv) insertionof internal ribosomal entry sites (IRESs).

In a preferred embodiment, multiple CAR units are expressed in a singleopen reading frame (ORF), thereby creating a single polypeptide havingmultiple CAR units. In this embodiment, an amino acid sequence or linkercontaining a high efficiency cleavage site is disposed between each CARunit.

As used herein, high cleavage efficiency is defined as more than 50%,more than 70%, more than 80%, or more than 90% of the translated proteinis cleaved. Cleavage efficiency may be measured by Western Blotanalysis, as described by Kim 2011.

Furthermore, in a preferred embodiment, there are equal amounts ofcleavage product, as shown on a Western Blot analysis.

Examples of high efficiency cleavage sites include porcine teschovirus-12A (P2A), FMDV 2A (abbreviated herein as F2A); equine rhinitis A virus(ERAV) 2A (E2A); and Thoseaasigna virus 2A (T2A), cytoplasmicpolyhedrosis virus 2A (BmCPV2A) and flacherie Virus 2A (BmIFV2A), or acombination thereof. In a preferred embodiment, the high efficiencycleavage site is P2A. High efficiency cleavage sites are described inKim J H, Lee S-R, Li L-H, Park H-J, Park J-H, Lee K Y, et al. (2011)High Cleavage Efficiency of a 2A Peptide Derived from PorcineTeschovirus-1 in Human Cell Lines, Zebrafish and Mice. PLoS ONE 6(4):e18556, the contents of which are incorporated herein by reference.

In embodiments wherein multiple CAR units are expressed in a single openreading frame (ORF), expression is under the control of a strongpromoter. Examples of strong promoters include the SFFV promoter, andderivatives thereof.

Engineered Cell Having CAR Polypeptide and Enhancer

In another embodiment, the present disclosure provides a method makingan engineered cell that expresses at least one CAR unit and an enhancer.

In some embodiments, at least one CAR unit and enhancer is expressed ina T or NK cell using bicistronic or multicistronic expression vectors.There are several strategies which can be employed to constructbicistronic or multicistronic vectors including, but not limited to, (1)multiple promoters fused to the CARs' open reading frames; (2) insertionof splicing signals between units of CAR; fusion of CARs whoseexpressions are driven by a single promoter; (3) insertion ofproteolytic cleavage sites between units of CAR (self-cleavage peptide);and (4) insertion of internal ribosomal entry sites (IRESs).

In a preferred embodiment, at least one CAR unit and an enhancer areexpressed in a single open reading frame (ORF), thereby creating asingle polypeptide having at least one CAR unit and an enhancer. In thisembodiment, an amino acid sequence or linker containing a highefficiency cleavage site is disposed between each CAR unit and between aCAR unit and enhancer. In this embodiment, the ORF is under the controlof a strong promoter. Examples of strong promoters include the SFFVpromoter, and derivatives thereof.

Furthermore, in a preferred embodiment, there are equal amounts ofcleavage product, as shown on a Western Blot analysis.

Methods of Treatment Using the Compositions Disclosed Herein

In another embodiment, the present disclosure provides a method oftargeting CD45 for conditioning prior to allogenic transplantation incancer treatment. CD45 is also known as leukocyte common antigen (LCA)and is a tyrosine phosphatase expressed on virtually all cells ofhematopoietic origin except erythrocytes and platelets. Most hematologicmalignancies express CD45. For instance, 85% to 90% acute lymphoid andmyeloid leukemias express CD45. CD45 is not found in non-hematopoieticorigin. In addition, CD45 is expressed at a high density of an averagecopy number of approximately 200,000 molecules per cells on malignantcells and leukocytes. CD45 presents an ideal target for a variety ofhematologic malignancies. However, CAR T and NK cells also express CD45.Without inactivation of endogenous CD45. CAR T or NK cells armed withCARs targeting CD45 may result in self-killing.

The association of CD45 with TCR complexes is essential in regulation ofT-cell activation in response to antigen. The inability ofCD45-deficient T cells to present antigen is due to reduced signalingthrough the T cell receptors (TCRs). TCRs are cell surface receptorsthat play an essential role in the activation of T cells in response tothe presentation of antigen. The TCR is generally made from two chains,alpha and beta, which are associated with the transducing subunits, theCD3, to form the T-cell receptor complex present on the cell surface.

It was surprisingly found that multiple CARs (Compound CARs, cCAR) ofthe present disclosure combat a key mechanism by which cancer cellsresist CAR activity, i.e., the downregulation or heterogeneousexpression of the target antigen from the cancer cell surface. Thismechanism allows the cancer cell to “hide” from the CAR therapy, aphenomenon referred to as ‘antigen escape’. The present disclosurepre-empts cancer antigen escape by recognizing a combination of two ormore antigens to rapidly eliminate the tumor.

The disclosure provides a method of simultaneous targeting ofmulti-antigens using a cCAR resulting in improved tumor control byminimizing the possibility of tumor selection on the basis of targetantigen loss or down-regulation.

The disclosed disclosure includes compound (multiple or compound) cCARin a T or NK cell targeting different or same surface antigens presentin tumor cells. The compound chimeric antigen receptors of the presentdisclosure comprise at least multiple chimeric receptor constructslinked by a linker and target of the same or different antigens. Forexample, each of the CAR construct present in the compound CAR (cCAR)construct includes an antigen recognition domain, an extracellulardomain, a transmembrane domain and/or a cytoplasmic domain. Theextracellular domain and transmembrane domain can be derived from anydesired source for such domains. The multiple CAR constructs are linkedby a linker. The expression of the compound CAR construct is driven by apromoter. The linker may be a peptide or a part of a protein, which isself-cleaved after a protein or peptide is generated (also called as aself-cleaving peptide).

In one embodiments, the compound CARs of the present disclosure targetMyelodysplastic Syndrome and acute myeloid leukemia (AML) populations.Myelodysplastic Syndrome (MDS) remains an incurable hematopoietic stemcell malignancy that occurs most frequently among the elderly, withabout 14,000 new cases each year in the USA. About 30-40% of MDS casesprogress to AML. The incidence of MDS continues to increase as ourpopulation ages. Although MDS and AML have been studied intensely, nosatisfactory treatments have been developed.

The compositions and methods of this disclosure can be used to generatea population of T lymphocyte or NK cells that deliver both primary andco-stimulatory signals for use in immunotherapy in the treatment ofcancer, in particular, the treatment of lung cancer, melanoma, breastcancer, prostate cancer, colon cancer, renal cell carcinoma, ovariancancer, brain cancer, sarcoma, leukemia and lymphoma.

Immunotherapeutics generally rely on the use of immune effector cellsand molecules to target and destroy cancer cells. The effector may be alymphocyte carrying a surface molecule that interacts, either directlyor indirectly, with a tumor cell target. Various effector cells includecytotoxic T cells, NK cells, and NK-92 cells. The compositions andmethods described in the present disclosure may be utilized inconjunction with other types of therapy for cancer, such aschemotherapy, surgery, radiation, gene therapy, and so forth. Thecompositions and methods described in the present disclosure may beutilized in other disease conditions that rely on immune responses suchas inflammation, immune diseases, and infectious diseases.

In some embodiments, the compound CAR of the present disclosure may actas a bridge to bone marrow transplant, by achieving complete remissionfor patients who have minimal residual disease and are no longerresponding to chemotherapy. In other embodiments, the compound CAReliminates leukemic cells followed by bone marrow stem cell rescue tosupport leukopenia.

In some embodiments, the compound CAR of the present disclosure cancombat a key mechanism by which cancer cells resist CAR activity by thedown-regulation of the target antigen. In another embodiment, theinvented compound CAR can also combat the heterogeneity of cancer cells,which creates significant challenges in a regular CAR T/NK cell therapy.In a further embodiment, the disclosed compound CAR is designed that thefirst CAR targets the bulky tumor population and another eradicatescancer or leukemic stem cells to avoid cancer relapses.

In one embodiment, the present disclosure provides a method ofdestroying cells having a CD33 antigen or a CD123 antigen, or both bycontacting said cells with an engineered cell having at least one ofchimeric antigen receptor polypeptide having a CD33 antigen recognitiondomain and chimeric antigen receptor polypeptide having a CD23 antigenrecognition domain. The engineered cell may be a T or NK cell.

Cells having at least one of the CD33 antigen and the CD123 antigeninclude acute myeloid leukemia, precursor acute lymphoblastic leukemia,chronic myeloproliferative neoplasms, chronic myeloid leukemia,myelodysplasia syndromes, blastic plasmocytoid dendritic neoplasms(BPDCN), Hodgkin's lymphoma, mastocytosis, and hairy cell leukemiacells.

In another embodiment, the present disclosure provides a method ofproviding myeloblative conditioning regimens for hematopoietic stem celltransplantation. In this embodiment, a T or NK engineered cell having aCD33 unit and a CD123 unit is administered to a patient in need thereof.

In further embodiments, the present disclosure provides a method oferadicating or killing leukemic stem cells (LSCs) or bulk leukemic cellsexpressing CD123 or CD33, or both. In this embodiment, a T or NKengineered cell having a CD33 unit and a CD123 unit is administered to apatient in need thereof.

In further embodiments, the compound CAR in a T or NK cell may be usedto eradicate or kill CD34+CD38− leukemic stem cells or bulk leukemiccells expressing CD123 or CD33 or both.

In some embodiments, a compound CAR targets cells expressing CD19 orCD20 antigens or both. In another embodiment, a compound CAR targetscells expressing CD19 or CD22 antigens or both. The targeted cells maybe cancer cells, such as, without limiting, B-cell lymphomas orleukemias. In further embodiments, the target antigens can include atleast one of this group, but not limited to, ROR1, PSMA, MAGE A3,Glycolipid, glypican 3, F77, GD-2, WT1, CEA, HER-2/neu, MAGE-3, MAGE-4,MAGE-5, MAGE-6, alpha-fetoprotein, CA 19-9, CA 72-4, NY-ESO, FAP, ErbB,c-Met, MART-1, CD30, EGFRvIII, immunoglobin kappa and lambda, CD38,CD52, CD3, CD4, CD8, CD5, CD7, CD2, and CD138. The target antigens canalso include viral or fungal antigens, such as E6 and E7 from the humanpapillomavirus (HPV) or EBV (Epstein Barr virus) antigens.

In some embodiments, the compound CAR engineered cells target cellshaving cell surface CD19 antigen or cell surface CD123 antigen or both.The targeted cells are cancer cells, such as, without limiting, B-celllymphomas or leukemias.

Clinical trials of CD19 CAR T cell therapy have shown that 80-94% ofpatients with B-ALL achieve complete remission, but a substantialproportion of patients eventually relapse. The prevalence of CD123expression in B-ALL is high, and can be used as a CAR target for B-ALL.

In some embodiments, the compound CAR targets cells expressing CD19 orCD123 antigen or both. Without wishing to be bound by theory, it isbelieved that CD19 and/or CD123 compound CAR engineered cells offsettumor escape due to the loss of CD19 or CD123 antigen or prevent B-ALLor other type B-cell lymphoma/leukemia relapse.

In further embodiments, the CD19 and/or CD20 compound CAR engineeredcells target cells having cell surface CD19 antigens and/or CD20 cellsurface antigens. In another embodiment, the targeted cells aremalignant B cell lymphoma/leukemia such as, without limiting, B-ALL,high grade B cell lymphoma, low grade B-cell lymphoma, diffuse large Bcell lymphoma, Burkett lymphoma, mantle cell lymphoma, CLL, marginalzone B cell lymphoma and follicular lymphoma.

Without wishing to be bound by theory, it is believed that the CD19and/or CD20 CAR engineered cells provide an effective safeguard againstantigen escape and prevent disease relapse in adoptive T/NK-cell therapyfor B-cell malignancies.

CAR target cells having at least one of the antigens CD19, CD20, CD22,and CD123, include precursor acute lymphoblastic leukemia, B-celllymphoma/leukemia, chronic lymphocytic leukemia/lymphoma, mantlelymphoma, follicular lymphoma, marginal zone B cell lymphoma, diffuselarge B cell lymphoma, Burkett lymphoma, blastic plasmocytoid dendriticneoplasms (BPDCN), Hodgkin's lymphoma, and hairy cell leukemia cells.

In one embodiment, the engineered cell includes a first chimeric antigenreceptor polypeptide having a CD19 antigen recognition domain and secondchimeric antigen receptor polypeptide having a CD22 antigen recognitiondomain. In one embodiment, this engineered cell includes a polypeptideof SEQ ID NO. 64 and corresponding polynucleotide of SEQ ID NO. 63.

In one embodiment, the engineered cell includes a first chimeric antigenreceptor polypeptide having a CD19 antigen recognition domain and secondchimeric antigen receptor polypeptide having a CD20 antigen recognitiondomain. In one embodiment, this engineered cell includes a polypeptideof SEQ ID NO. 66 and corresponding polynucleotide of SEQ ID NO. 65.

In one embodiment, the engineered cell includes a first chimeric antigenreceptor polypeptide having a CD19 antigen recognition domain and secondchimeric antigen receptor polypeptide having a CD123 antigen recognitiondomain. In one embodiment, this engineered cell includes a polypeptideof SEQ ID NO. 68 and corresponding polynucleotide of SEQ ID NO. 67.

Multiple myeloma is an incurable disease exhibiting uncontrollableproliferation of plasma cells in the bone marrow. CS1 and BCMA arewidely expressed on myeloma cells, but is not expressed in hematopoieticstem/progenitor cells. Therefore, BCMA and CS1 are ideal targets for CART/NK cell therapy.

In further embodiments, the present disclosure provides compound CARengineered cell having a CS1 (SLAM7) antigen recognition domain and/oran antigen recognition domain that targets B-cell maturation antigens(BCMA). In another embodiment, the targeted cells are malignant plasmacells, such as, but not limited to, multiple myeloma.

Without wishing to be bound by theory, it is believed that a compoundCAR engineered cell having at least one of CS1 and BCMA antigenrecognition domain enhances functionality against multiple myeloma andoffset antigen escape.

In some embodiments, a CAR targets cells expressing multiple antigensincluding, but not limited to, CS1, BCMA, CD267, BAFF-R, CD38, CD138,CD52, CD19, TACI, CD20, interleukin 6 receptor, and NY-ESO-1 antigens.In another embodiment, the targeted cells are plasma cells, B-cells,malignant plasma cells such as, without limiting, multiple myeloma.

In some embodiments, the compound CAR targets cells expressing multipleantigens including, but not limited to, CS1, BCMA, CD267, BAFF-R, CD38,CD138, CD52, CD19, TACI, CD20, interleukin 6 receptor, and NY-ESO-1antigens. In another embodiment, the targeted cells are malignant plasmacells such as, without limiting, multiple myeloma.

In some embodiments, the compound CAR targets cells expressing multipleantigens including but not limited to, alpha fetoprotein (AFP) andGlypican-3 (GPC3). In another embodiment, the targeting cells arehepatocellular carcinoma, fibrolamellar carcinoma, hepatoblastoma,undifferentiated embryonal sarcoma and mesenchymal hamartoma of liver,lung-squamous cell carcinoma, testicular nonseminomatous germ celltumors, liposarcoma, ovarian and extragonadal yolk sac tumors, ovarianchoriocarcinoma, teratomas, ovarian clear cell carcinoma, and placentalsite trophoblastic tumor.

Without wishing to be bound by theory, the present disclosure providescompound CAR engineered T cells or NK cells that target different or thesame antigens offset tumor escape and provides simultaneous targeting oftumor cells.

The T or NK host cells comprising compound CAR disclosed herein isembodied in the present disclosure. The nucleotide and polypeptideconstructs, sequences, host cells, and vectors of the compound CAR areconsidered to be part of the present disclosure and is embodied herein.

In some embodiments, the compound CAR engineered cell is administratedin combination with any chemotherapy agents currently being developed oravailable in the market. In some embodiments, the compound CARengineered cell is administrated as a first line treatment for diseasesincluding, but not limited to, hematologic malignancies, cancers,non-hematologic malignances, inflammatory diseases, infectious diseasessuch as HIV and HTLV and others. In one embodiment, T cells expressingthe compound CAR engineered cells are co-administrated with NK cellsexpressing the same or different compound CAR as an adaptiveimmunotherapy. Compound CAR NK cells provide rapid, innate activitytargeting cells while compound T cells provide relative long-lastingadaptive immune activity.

In one embodiment, the compound CAR engineered cells are administratedas a bridge to bone marrow stem transplantation for mammals, e.g.patients who are resistant to chemotherapies and are not qualified forbone marrow stem cell transplantation.

In some embodiments, the compound CAR co-expresses a transgene andreleases a transgenic product, such as IL-12 in the targeted tumorlesion and further modulates the tumor microenvironment.

In one embodiment, compound CAR engineered cells are administrated to amammal for bone marrow myeloid ablation as a part of the treatment to adisease.

In a specific embodiment, the cells expressing a compound CAR can be Tcells or NK cells, administrated to a mammal, e.g. human. The presenteddisclosure includes a method of treating a mammal having a disorder ordisease by administration of a compound CAR. The targeted cells may becancer cells such as, or cells affected by any other disease condition,such as infectious diseases, inflammation, and autoimmune disorders.

The present disclosure is intended to include the use of fragments,mutants, or variants (e.g., modified forms) of the compound CAR orantigens that retain the ability to induce stimulation and proliferationof T/NK cells. A “form of the protein” is intended to mean a proteinthat shares a significant homology with at least one CAR or antigen andis capable of effecting stimulation and proliferation of T/NK cells. Theterms “biologically active” or “biologically active form of theprotein,” as used herein, are meant to include forms of the proteins orvariants that are capable of effecting anti-tumor activity of the cells.

The compositions and methods of this disclosure can be used to generatea population of T/NK cells that deliver both primary and co-stimulatorysignals for use in immunotherapy in the treatment of cancer, inparticular the treatment of lung cancer, melanoma, breast cancer,prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer,neuroblastoma, rhabdomyosarcoma, leukemia and lymphoma. The compositionsand methods described in the present disclosure may be utilized inconjunction with other types of therapy for cancer, such aschemotherapy, surgery, radiation, gene therapy, and so forth.

-   -   1) In some embodiments, the disclosure provides a method of        depleting B cells, immature B cells, memory B cells,        plasmablasts, long lived plasma cells, or plasma cells in        patients with an autoimmune disease by administering to patients        CAR or compound CAR IT cells or NK cells. CAR targeted cells are        B or plasma cells expressing one or two or all of the antigens,        BCMA, TACI and BAFF-R. The autoimmune diseases include systemic        scleroderma, multiple sclerosis, psoriasis, dermatitis,        inflammatory bowel diseases (such as Crohn's disease and        ulcerative colitis), systemic lupus erythematosus, vasculitis,        rheumatoid arthritis, Sjorgen's syndrome, polymyositis,        pulmonary alveolar proteinosis, granulomatosis and vasculitis.        Addison's disease, antigen-antibody complex mediated diseases,        and anti-glomerular basement membrane disease.

Multiple extracellular cell markers are now being studied for value astumor-associated antigens and thus potential targets for CAR T/NK celltherapy. However, expression of these antigens on healthy tissue leadingto on-target, off-tumor adverse events remains a major safety concern inaddition to off-target toxicities. Furthermore, a major limitation ofCAR T/NK cell therapy is in the possibility of selecting for antigenescape variants when targeting molecules non-essential to tumorigenesis.Thus, malignant cells that persist with little or no expression of thetarget antigens may evade CAR T/NK cells, despite their high-affinityaction.

In accordance with the present disclosure, natural killer (NK) cellsrepresent alternative cytotoxic effectors for CAR driven killing. UnlikeT-cells, NK cells do not need pre-activation and constitutively exhibitcytolytic functions. Further expression of cCARs in NK cells allow NKcells to effectively kill cancers, particularly cancer cells that areresistant to NK cell treatment.

Further, NK cells are known to mediate anti-cancer effects without therisk of inducing graft-versus-host disease (GvHD).

Studies have shown an aberrant overexpression of CD123 on CD34+CD38− AMLcells, while the normal bone marrow counterpart CD34+CD38− does notexpress CD123 (Jordan, Upchurch et al. 2000). This population of CD123+,CD34+CD38− has been considered as LSCs as these cells are able toinitiate and maintain the leukemic process in immunodeficient mice.

The number of CD34+/CD38−/CD123+ LSCs can be used to predict theclinical outcome for AML patients. The CD34+/CD38−/CD123+ cells, greaterthan 15% in AML patients, are associated with a lack of completeremission and unfavorable cytogenetic profiles. In addition, thepresence of more than 1% of CD34+/CD38−/CD123+ cells could also have anegative impact on disease-free survival and overall survival.

At the present, therapies for MDS and AML have focused on the leukemicblast cells because they are very abundant and clearly represent themost immediate problem for patients. Importantly, leukemic stem cells(LSCs) are quite different from most of the other leukemia cells(“blast” cells), and they constitute a rare subpopulation. While killingblast cells can provide short-term relief, LSCs, if not destroyed, willalways re-grow, causing the patient to relapse. It is imperative thatLSCs be destroyed in order to achieve durable cures for MDS disease.Unfortunately, standard drug regimens are not effective against MDS orAML LSCs. Therefore, it is critical to develop of new therapies that canspecifically target both the leukemic stem cell population and the bulkyleukemic population. The compound CAR disclosed in the presentdisclosure target both of these populations and is embodied herein.

In accordance to the present disclosure, it was surprisingly found thatNK cells provide an off-the-shelf product that may be used as anallogeneic product for treatment. Thus, according to the presentdisclosure, cCAR cell therapy needs to be performed on apatient-specific basis as required by the current state of art. Theapplicants of the present disclosure have discovered a novelimmunotherapy, where the patient's lymphocytes or tumor infiltratedlymphocytes need not be isolated for an effective CAR cell basedtherapy.

Allogeneic or autologous NK cells are expected to induce a rapid immuneresponse but disappear relatively rapidly from the circulation due totheir limited lifespan. Thus, applicants surprisingly discovered thatthere is reduced concern of persisting side effects using cCAR cellbased therapy.

According to one aspect of the present disclosure, NK cells can beexpanded and transfected with cCAR in accordance to the presentdisclosure. NK cells can be derived from cord blood, peripheral blood,iPS cells and embryonic stem cells. According to one aspect of thepresent disclosure, NK-92 cells may be expanded and transfected withcCAR. NK-92 is a continuously growing cell line that has features andcharacteristics of natural killer (NK) cells. NK-92 cell line is IL-2dependent and has been proven to be safe and feasible. cCAR expressingNK-92 cells can be expanded in the serum free-medium with or withoutco-culturing with feeder cells. A pure population of NK-92 carrying thecCAR of interest may be obtained by sorting.

Identification of appropriate surface target antigens is a prerequisitefor developing CAR T/NK cells in adaptive immune therapy.

In one aspect of the present disclosure, CD123 antigen is one of thetargets for cCAR therapy. CD123, the alpha chain of the interleukin 3receptor, is overexpressed on a variety of hematologic malignancies,including acute myeloid leukemia (AML), B-cell acute lymphoblasticleukemia (B-ALL), hairy cell leukemia, and blastic plasmocytoiddendritic neoplasms. CD123 is absent or minimally expressed on normalhematopoietic stem cells. More importantly, CD123 is expressed on asubset of leukemic cells related to leukemic stem cells (LSCs), theablation of which is essential in preventing disease refractoriness andrelapse.

In one aspect of the present disclosure, CD 33 antigen is one of thetargets for cCAR therapy. CD33 is a transmembrane receptor expressed on90% of malignant cells in acute myeloid leukemia. Thus, according to thepresent disclosure, CD123 and CD33 target antigens are particularlyattractive from a safety standpoint.

In accordance with the present disclosure, the compound CD33CD123 CARsmay be highly effective for therapeutic treatment of chronic myeloidleukemia (CML) population. In chronic myeloid leukemia (CML), there is arare subset of cells that are CD34+CD38−. This population is consideredas comprised of LSCs. Increased number of LSCs is associated with theprogression of the disease. A small-molecule Bcr-Abl tyrosine kinaseinhibitor (TKI) is shown to significantly improve the overall survivalin CP-CML patients. However, LSCs are thought to be resistant to TKItherapy. A novel therapy targeting CML resistant LSCs is urgently neededfor treatment of CML and the novel therapy is embodied in the compoundCD33CD123 CAR disclosed in the present disclosure. CD123 expression ishigh in the CD34+CD38-population. In accordance with the presentdisclosure, the compound CD33CD123 CARs is highly effective fortherapeutic treatment of this population.

In one embodiment of the present disclosure, leukemic cells expressingboth CD123 and CD33 in the cCAR is used as a therapeutic treatment. CD33is expressed on cells of myeloid lineage, myeloid leukemic blasts, andmature monocytes but not normal pluripotent hematopoietic stem cells.CD33 is widely expressed in leukemic cells in CML, myeloproliferativeneoplasms, and MDS.

Since a significant number of patients with acute myeloid leukemia (AML)are refractory to standard chemotherapy regimens or experience diseaserelapse following treatment (Burnett 2012), the development of CAR Tcell immunotherapy for AML has the potential to address a great clinicalneed. In the majority of these patients, leukemic cells express bothCD123 and CD33, giving broad clinical applicability to the compoundCD33CD123 CAR disclosed herein. Thus, the present disclosure discloses anovel multiple cCAR T/NK cell construct comprising multiple CARstargeting multiple leukemia-associated antigens, thereby offsettingantigen escape mechanism, targeting leukemia cells, including leukemicstem cells, by synergistic effects of co-stimulatory domain activation,thereby providing a more potent, safe and effective therapy.

The present disclosure further discloses a compound CAR construct withenhanced potency of anti-tumor activity against cells co-expressingtarget antigens, and yet retains sensitivity to tumor cells onlyexpressing one antigen. In addition, each CAR of the compound CARincludes one or two co-stimulatory domains and exhibits potent killingcapability in the presence of the specific target.

In pre-clinical studies on dual specificity, trans-signaling CARstargeting solid tumors including breast cancer and epithelial ovariancancer, a CD3ζ intracellular signaling domain, is separated fromco-stimulatory domains from second generation of CARs. In other words,one CAR contains the first generation of CAR without any co-stimulatorydomain, and another lacks a CD3 zeta intracellular domain. Therefore,the presence of both target antigens is required for T cell activationand potent killing. Thus, they were proposed as a way to decreaseoff-tumor toxicity caused by healthy tissue expression of one of the twotarget antigens, increasing target specificity, but at the expense ofsensitivity. In one embodiment, the compound CAR is a compound CD123CD19CAR. It has been shown that more than 90% of B-ALLs express CD123 in asubset of population. Like AML and MDS, it has been considered that arare LSC population exists in B-ALL. Therefore, targeting both leukemicstem cell and bulky leukemic populations in accordance to the presentdisclosure, can be applied to B-ALLs. CD123 and CD19 surface antigensexpressed in the B-ALLs may be targets as CD19 is widely expressed indifferent stages of B-cell lymphoid populations, in accordance with thepresent disclosure.

Multiple myeloma (MM) is the second most common hematologic malignancyin the US and is derived from clonal plasma cells accumulated in thebone marrow or extramedullary sites. MM is an incurable disease with amedian survival of approximately 4.5 years. Anti-Myeloma CARs inPre-clinical Development have been developed and CAR targets includeCD38, CS1, and B cell maturation Antigen (BCMA). However, heterogeneityof surface antigen expression commonly occurs in malignant plasma cells,which makes it a difficult target for CARs. Malignant plasma cells alsoexpress low levels of CD19. Previously it has been shown that myelomastem cells also express some B-cell markers including CD19. Targetingthis population could be effective in the treatment of myeloma inconjunction with standard and other myeloma CAR therapies.

Multiple myeloma (MM) is a haematological malignancy with a clonalexpansion of plasma cells. Despite important advances in the treatment,myeloma remains an incurable disease; thus novel therapeutic approachesare urgently needed.

CS1 (also called as CD319 or SLAMF7) is a protein encoded by the SLAMF7gene. The surface antigen CS1 is a robust marker for normal plasma cellsand myeloma cells (malignant plasma cells).

Tumour necrosis factor receptor superfamily, member 17 (TNFRSF17), alsoreferred to as B-cell maturation antigen (BCMA) or CD269 is almostexclusively expressed at the terminal stages of plasma cells andmalignant plasma cells. Its expression is absent other tissues,indicating the potential as a target for CAR T or NK cells.

Malignant plasma cells display variable degrees of antigenicheterogeneity for CD269 and CS1. A single CAR unit product targetingeither CD269 or CS1 could target the majority of the cells in a bulktumor resulting in an initial robust anti-tumor response. Subsequentlyresidual rare non-targeted cells are expanded and cause a diseaserelapse. While multiple myeloma is particularly heterogeneous, thisphenomena could certainty apply to other leukemias or tumors.

A recent clinical trial at NIH using BCMA CAR T cells showed a promisingresult with a complete response in some patients with multiple myeloma.However, these patients relapsed after 17 weeks, which may be due to theantigen escape. The antigen escape is also seen in CD19 CAR and NY-ESO1CAR T cell treatments. Thus, there is an urgent need for more effectiveCAR T cell treatment in order to prevent the relapse.

In one aspect of the present disclosure, BCMA and CS1 are the targetsfor BCMACS1 CAR therapy.

In some embodiments, a compound CAR targets cells expressing BCMA or CS1antigens or both. The targeted cells may be cancer cells, such as,without limiting, lymphomas, or leukemias or plasma cell neoplasms. Infurther embodiments, plasma cell neoplasms is selected from plasma cellleukemia, multiple myeloma, plasmacytoma, heavy chain diseases,amyloidosis, waldestrom's macroglobulinema, heavy chain diseases,solitary bone plasmacytoma, monoclonal gammopathy of undeterminedsignificance (MGUS) and smoldering multiple myeloma.

In some embodiments, the present disclosure provides a compound CARpolypeptide engineered cell that targets cells expressing BCMA or CD19antigens or both. The targeted cells may be cancer cells, such as, butnot limited to, lymphomas, or leukemias or plasma cell neoplasms. Infurther embodiments, plasma cell neoplasms are selected from plasma cellleukemia, multiple myeloma, plasmacytoma, heavy chain diseases,amyloidosis, waldestrom's macroglobulinema, heavy chain diseases,solitary bone plamacytoma, monoclonal gammopathy of undeterminedsignificance (MGUS) and smoldering multiple myeloma.

BAFF (B-cell-activation factor) and APRIL (a proliferation-inducedligand) are two TNF homologs that bind specifically TACI (also called asTNFRSF1 3B or CD267) and BCMA with high affinity. BAFF (also known asBLyS) binds BAFF-R and functionally involves in the enhancement ofsurvival and proliferation of later stage of B cells. BAFF has beenshown to involve some autoimmune disorders. APRIL plays an importantrole in the enhancement of antibody class switching. Both BAFF and APRILhave been implicated as growth and survival factors for malignant plasmacells.

Ligand-receptor interactions in the malignant plasma cells or normalplasma cells are described in FIGS. 77 and 79 .

In some embodiments, the present disclosure provides a compound CARengineered cell that targets cells expressing TACI or CS1 antigens orboth. In another embodiment, a compound CAR engineered cell that targetscells expressing TACI or CS1 antigens or both. The targeted cells may becancer cells, such as, without limiting, lymphomas, or leukemias orplasma cell neoplasms. In further embodiments, plasma cell neoplasms isselected from plasma cell leukemia, multiple myeloma, plasmacytoma,heavy chain diseases, amyloidosis, waldestrom's macroglobulinema, heavychain diseases, solitary bone plamacytoma, monoclonal gammopathy ofundetermined significance (MGUS) and smoldering multiple myeloma. Thetarget cells may also be one or two or multiple different cell types ofB cells, immature B cells, naïve B cells, centroblasts, centrocytes,memory B cells, plasmablasts, long lived plasma cells, plasma cells.These cells involve autoimmune diseases include systemic scleroderma,multiple sclerosis, psoriasis, dermatitis, inflammatory bowel diseases(such as Crohn's disease and ulcerative colitis), systemic lupuserythematosus, vasculitis, rheumatoid arthritis, Sjorgen's syndrome,polymyositis, granulomatosis and vasculitis, Addison's disease,antigen-antibody complex mediated diseases, and anti-glomerular basementmembrane disease.

In some embodiments, the present disclosure provides a compound CARengineered cell that targets cells expressing BAFF-R or CS1 antigens orboth. In another embodiment, a compound CAR engineered cell that targetscells expressing BAFF-R or CS1 antigens or both. The targeted cells maybe cancer cells, such as, without limiting, lymphomas, or leukemias orplasma cell neoplasms. In further embodiments, plasma cell neoplasms isselected from plasma cell leukemia, multiple myeloma, plasmacytoma,heavy chain diseases, amyloidosis, waldestrom's macroglobulinema, heavychain diseases, solitary bone plamacytoma, monoclonal gammopathy ofundetermined significance (MGUS) and smoldering multiple myeloma.

Autoimmune disorders such as lupus erythematosus (SLE), pemphigusvulgaris and multiple sclerosis (MS) cause significant morbidity anddisability. These diseases respond poorly to current therapies andfrequent relapse during disease course is noted. B and plasma cells playa critical role in the pathogenesis of autoimmune disorders as they arethe sources of autoantibody production. 13 and plasma cells maycontribute to disease progression and relapse through antigenpresentation, cytokine secretion, or antibody production. Deletion of Bcells or plasma cells or both using CAR T/NK cell approaches can be abeneficial therapy.

An organ transplant represents a new life for a person and organs thatcan be transplanted could include the kidneys, heart, lungs, pancreasand intestine. However, many patients are unable to receive apotentially life-saving organ because of pre-existing or developingdonor-specific antibody against the donor's antigens such humanleukocyte antigens (HLA). Thus, patients may lose the donated organ.Currently there are few treatment options available for antibodymediated rejection, and an enormous unmet need in the field forefficacious treatment of antibody mediated rejection. Deletion of Bcells or plasma cells or both using CAR T/NK cell provide a therapy forantibody-mediated rejection.

The disclosed disclosure provides compositions and methods relating toCAR engineered cells that target cells expressing CD19 or CD20 thatresult in the deletion of B cells but spare long-lived plasma cells inpatients with antibody mediated organ rejection or autoimmune disordersincluding, but not limited to, systemic lupus erythematosus (SLE),rheumatoid arthritis (RA), and pemphigus vulgaris and multiple sclerosis(MS). The deletion of B cells by CAR is beneficial to the reduction ofdisease activity.

The present disclosure also provides compositions and methods relatingto CAR engineered cells that target cells expressing BCMA or BAFF-R,TACI which results in the deletion of plasma cells in patients withantibody mediated organ rejection or autoiminune disorders including,but not limited to, systemic lupus erythematosus (SLE), rheumatoidarthritis (RA), and pemphigus vulgaris and multiple sclerosis (MS). Thedeletion of plasma cells can contribute to the reduction of diseaseactivity.

In some embodiments, the present disclosure provides compositions andmethods relating to CAR engineered cells for CARs depleting mature,memory B cells, and short, long lived plasma cells for treatment ofautoimmune disorders and organ antibody-mediated organ rejection. In oneembodiment, the present disclosure provides a method for depletingmature, memory B cells, and short, long lived plasma cells using one ormore of the following strategies:

-   -   1) Depletion of mature, memory B cells and short, long lived        plasma cells by a contacting said cells with an CAR engineered        cell having a scFv against CD19 or CD20 or CD22:    -   2) Depletion of short- and long-lived plasma cells by contacting        said cells with a CAR engineered cell having a seFv against BCMA        or TACI or BAFF-R; or    -   3) Depletion of short- and long-lived plasma cells by contacting        said cells with a CAR engineered cell having an antigen        recognition domain including BCMA or TACI or BAFF-R binding        domain (BAFF or APRIL);    -   4) Deletion of mature, memory B cells, and short, long lived        plasma cells contacting said cells with a compound CAR        engineered cell targeting more than one different antigen to        provide a reduction of disease activity for patients with        antibody mediated organ rejection or autoimmune disorders.    -   5) Deletion of mature, memory B cells, and short, long lived        plasma cells by contacting a CAR engineered cells that target        more than one different antigen selecting from CD19, CD20,        (CD22, B3CMA, TACI, APRIL and BAFF.

In some embodiments, a compound CAR (cCAR) targets cells expressing oneor two or all of BAFF-R, BCMA, TACI and CS1 antigens.

In some embodiments, a unit of CAR in a cCAR can comprise: 1) a scFvagainst either BAFF-R, BCMA, TACI and CS1; 2) a hinge region; 3)co-stimulatory domain (s) and intracellular signaling domain.

In some embodiments, BAFF CAR can be a unit of CAR in a cCARcomprises: 1) BCMA or TACI or BAFF-R binding domain; 2) a hinge region;3) co-stimulatory domain (s) and intracellular signaling domain.

In some embodiments, APRIL CAR can be a unit of CAR in a cCARcomprises: 1) BCMA or TACI binding domain; 2) a hinge region; 3)co-stimulatory domain (s) and intracellular signaling domain.

In a further embodiment, BCMA or TAC1 or BAFF-R binding domain can be apart of or entire APRIL and BAFF molecules.

In some embodiments, a unit of CAR in a cCAR can comprise: 1) a scFvagainst BCMA or CS1; 2) a hinge region; 3) co-stimulatory domain (s) andintracellular signaling domain.

In some embodiments, a unit of CAR in a cCAR can comprise: 1) a scFvagainst BCMA or CD19; 2) a hinge region; 3) co-stimulatory domain (s)and intracellular signaling domain.

In some embodiments, a unit of CAR in a cCAR can comprise: 1) a scFvagainst BCMA or CD20; 2) a hinge region; 3) co-stimulatory domain (s)and intracellular signaling domain.

In some embodiments, a unit of CAR in a cCAR can comprise: 1) BAFF-Rbinding domain or a scFv against BCMA; 2) a hinge region; 3)co-stimulatory domain (s) and intracellular signaling domain.

In some embodiments, a unit of CAR in a cCAR can comprise: 1) BAFF-Rbinding domain or a scFv against CD19; 2) a hinge region; 3)co-stimulatory domain (s) and intracellular 20 signaling domain.

In some embodiments, a unit of CAR in a cCAR can comprise: 1) BAFF-Rbinding domain or a scFv against CD20; 2) a hinge region; 3)co-stimulatory domain (s) and intracellular signaling domain.

It is unexpected that some myeloma cells are dim (weak) or negative forBCMA. To increase the sensitivity of CAR antigen recognition in myelomacells, it is critical to target multiple antigens to cure this disease.

TACI, BCMA and BAFF-R are receptors for BAFF. It is also unexpected thatsome myeloma cells express TACI or BAFF-R over BCMA.

In some embodiments, the disclosure provides a method of comprising aBAFF CAR targeting a cell expressing at least one of receptors, BAFF-R,TACI and BCMA to improve therapeutic efficacy and reduce the risk ofantigen disease escape.

The affinity for BAFF binding to BCMA is within the micromolar range,which is much lower than that of BAFF-R or TACI in the nanomolar range.

In some embodiments, the disclosure provides a method of generating acompound cCAR comprising BAFF and BCMA CARs to complement some ofmyeloma cells that cannot be eliminated by a BAFF CAR.

In further embodiments, cCAR can comprise one or two or multiple unitsof CAR. Each unit CAR could bear same or different hinge region andco-stimulatory domain.

In further embodiments, cCAR polypeptides two or more CAR polypeptideunits. Each unit CAR could bear a different polynucleotide sequence toavoid a homologous recombination.

In some embodiments, targeting more than one different antigen can beachieved by pooled CAR engineered cells, which are generated by at leasttwo separate CAR T or NK cells.

As used herein, pooled CAR engineered cells include a population ofengineered cells having more than one distinct CAR polypeptide unit. Byway of example, pooled engineered cells include a population ofengineered cells with a distinct CAR polypeptide and a population ofengineered cells with a different and distinct CAR polypeptide.Furthermore, the pooled CAR engineered cells include engineered cellshaving cCAR polypeptides.

The pooled CAR T or NK cells can be completed by the following steps:

-   -   1) Generate at least two separate constructs of CARs targeting        different antigens; 2) Transduce individual construct to T or NK        cells and expand them ex vivo in a standard medium;    -   3) Pool different expanded T or NK cells at an appropriate        ratio; and    -   4) Administer pooled CAR T or NK cells to a subject.

In the alternative, the different engineered cells may be individualexpanded and independently or sequentially administered.

In further embodiments, the target antigens can include at least one ofthis group, but not limited to, ROR1, PSMA, MAGE A3, Glycolipid,glypican 3, F77, GD-2, WT1, CEA, HER-2/neu, MAGE-3, MAGE-4, MAGE-5,MAGE-6, alpha-fetoprotein, CA 19-9, CA 72-4, NY-ESO, FAP, ErbB, c-Met,MART-1, CD30, EGFRvIII, immunoglobin kappa and lambda, CD38, CD52, CD3,CD4, CD8, CD5, CD7, CD2, and CD138. The target antigens can also includeviral or fungal antigens, such as E6 and E7 from the humanpapillomavirus (HPV) or EBV (Epstein Barr virus) antigens.

In some embodiments, a cCAR engineered cell targets a cell expressingeither CD19 or CD20 antigens or both of them. In another embedment, acCAR engineered cells target a cell expressing either CD19 or CD22antigens or both of them. The targeted cells are normal B cellsassociated with autoimmune disorders or cancer cells such as B-celllymphomas or leukemias.

Acute graft-versus-host disease (GVHD) remains the most important causeof morbidity and mortality after allogeneic hematopoietic stem celltransplantation. In the effector phase of GVHD, T cell receptor (TCR), aheterodimer of alpha and beta chains, is expressed on the surface of Tcells, TCR recognizes some antigens on the HLA molecule on host cells,enhances T cell proliferation, and releases cytotoxic agents that causethe damage on host cells. TCR gene inactivation is efficient atpreventing potential graft-versus-host reaction. The inactivation ofTCRs can result in the prevention of the TCR recognition of alloantigenand thus GVHD. The role of CD45 on NK cells is quite different from thatof T cells. NK cells from CD45-deficient mice have normal cytotoxicactivity against the prototypic tumor cell line, Yac-1. In addition,CD45-deficient NK cells proliferate normally and respond to IL-15 andIL-21. Therefore, CD45 disruption or deletion would not affect the NKcell killing and proliferation. The present disclosure includes methodsof permanent deletion of CD45 in a T or NK cell with subsequent stableintroduction of CD45-specific CARs. As a result, the engineered T cellsdisplay the desired properties of redirected specificity for CD45without causing self-killing and response to presentation of antigen. Ina further embodiment, the engineered T cells may have efficacy as anoff-the-shelf therapy for treating malignancies or other diseases. Thepresent disclosure relates to a method where T-cells are engineered toallow proliferation when TCR signaling is reduced or lost through theinactivation or deletion of endogenous CD45. The reduction or loss ofTCR signaling could result in the prevention of GVHD. In a furtherembodiment, T cells reducing or losing the TCR signaling by theinactivation of CD45 could be used as an “off the shelf” therapeuticproduct.

The present disclosure includes methods of modified T or NK cells, whichcomprises: (a) modifying T or NK cells by inactivating CD45; (b)expanding these modified cells; (c) sorting modified T or NK cells,which do not express CD45; (d) introducing CD45CAR. In embodiments, theCD45CAR gene encodes a chimeric antigen receptor (CAR), wherein the CARcomprises at least one of an antigen recognition domain, a hinge region,a transmembrane domain, and T cell activation domains, and the antigenrecognition domain is redirected against CD45 surface antigen present ona cell. The antigen recognition domain includes a monoclonal antibody ora polyclonal antibody directed against CD45 antigen. The antigenrecognition domain includes the binding portion or a variable region ofa monoclonal or a polyclonal antibody.

The present disclosure includes methods of improving CD45CAR T/NK cellexpansion, persistency and anti-tumor activity by co-expressingsecretory IL-15/IL-15sushi complexes. In a further embodiment,engineered CD45CAR T/NK cells comprise secretory IL-15/IL-15sushicomplexes, which can promote expansion of specific CAR T/NK cells, andpromote infiltrate of innate immune cells to the tumor sites resultingin tumor destruction.

The present disclosure provides an alternative strategy in whichengineered CD45 CAR T cells receive not only costimulation through theCD28 pathway but also co-express the 4-1BB ligand (CD137L), whichprovide high therapeutic efficacy.

In some embodiments, the modified T cells are obtained from allogeneicdonors and used as an “off-the-shelf product”.

Targeting CD45 using CAR T or NK cells may cause self-killing as T andNK cells express this surface antigen. To overcome this drawback, thepresent disclosure provides engineered cells that are deficient in CD45.As used herein, an engineered cell is deficient for a particular genewhen expression of the gene is reduced or eliminated. Reduction orelimination of expression can be accomplished by any genetic methodknown in the art. In one example, the CD45 gene may be inactivated byusing engineered CRISPR/Cas9 system, zinc finger nuclease (ZFNs) andTALE nucleases (TALENs) and meganucleases. The loss of CD45 in T or NKcells is further transduced with CARs targeting neoplasms expressingCD45.

The disclosure includes methods for eliminating or reducing abnormal ormalignant cells in bone marrow, blood and organs. In, B and someembodiments, malignant cells expressing CD45 are present in patientswith acute leukemia, chronic leukemia T cell lymphomas, myeloidleukemia, Acute lymphoblastic lymphoma or leukemia, primary effusionlymphoma, Reticulohistiocytoma, transient myeloproliferative disorder ofDown's syndrome, lymphocyte predominant Hodgkin's lymphoma, myeloidleukemia or sarcoma, dendrocytoma, histiocytic sarcoma, Giant cell tumorof tendon sheath, interdigitating dendritic cell sarcoma,post-transplant lymphoproliferative disorders, etc.

Hematopoietic stem cell transplantation (HSCT) has been widely used forthe treatment of hematologic malignancies or non-hematologic diseases.Non-hematologic diseases include genetic disorders andimmunodeficiencies and autoimmune disorders. Genetic disorders include,not limited to, sickle cell disease, thalassemia and lysosomal storagediseases. Before stem cell transplant, patients are required to undergoa conditional therapy to deplete hematopoietic stem/progenitor cells inthe bone marrow niches to promote the donor stem cell engraftment andproliferation. High doses of chemotherapies and total body irradiationare used for conditional therapies, which cause severe toxicity andlong-term complications particularly in non-hematopoietic organs such assevere intestinal mucositis. In addition, conventional conditionaltherapies could destruct bone marrow niches resulting hematopoietic cellrecovery. The safety concerns represent a major obstacle in effort toexpand HSCT to a variety of non-hematologic diseases. CD45 is expressedonly on hematopoietic cells and targeting CD45 should minimize thetoxicity to non-hematopoietic organs.

In some embodiments, CD45CAR cells are used to make space in the bonemarrow for bone marrow stem cell transplant by removing hematopoieticcells, at the same time removing leukemic/lymphoma cells or immunologiccells capable of graft rejection.

In some embodiments, CD45CAR engineered cells are used to depletehematopoietic stem/progenitor cells while the architecture andvasculature of the bone marrow are preserved, in contrast to thedisruptive effects of total body irradiation on these tissues.Preservation of the architecture and vasculature of the bone marrowallows faster hematopoietic recovery after transient CD45CAR treatment.

In a further embodiment, CD45CAR cells may be used for pre-treatment ofpatients before their undergoing a bone marrow transplant to receivestem cells. In a further embodiment, CD45CAR can be used as myeloblativeconditioning regimens for hematopoietic stem cell transplantation.

In a preferred embedment, CD45CAR engineered cell therapy is transientfor allowing faster recovery of bone marrow and peripheral hematopoieticcells. Transient therapy may be accomplished by using short livedengineered cells or providing an engineered cell having the suicidesystem as described herein.

In some embodiments, the present disclosure comprises a method ofselectively depleting or ablating an endogenous hematopoieticstem/progenitor population, where the endogenous hematopoieticstem/progenitor cells expressing CD45, by contacting said cells withCD45CAR engineered cell that specifically targets CD45 expressinghematopoietic stem/progenitor cells.

In some embodiment, CD45CAR cells are utilized for treating orpreventing a residual disease after stem cell transplant and/orchemotherapy.

In some embodiments, the CD45CAR is part of an expressing gene or acassette. In a preferred embodiment, the expressing gene or the cassetteincludes an accessory gene or a tag or a part thereof, in addition tothe CD45CAR. The accessory gene may be an inducible suicide gene or apart thereof, including, but not limited to, caspase 9 gene, thymidinekinase, cytosine deaminase (CD) or cytochrome P450. The “suicide gene”ablation approach improves safety of the gene therapy and kills cellsonly when activated by a specific compound or a molecule. In someembodiments, the suicide gene is inducible and is activated using aspecific chemical inducer of dimerization (CID).

In some embodiments, the safety switch can include the accessory tagsare a c-myc tag, CD20, CD52 (Campath), truncated EGFR gene (EGFRt) or apart or a combination thereof. The accessory tag may be used as anonimmunogenic selection tool or for tracking markers. In someembodiments, safety switch can include a 24-residue peptide thatcorresponds to residues 254-277 of the RSV F glycoprotein A2 strain(NSELLSLINDMPITNDQKKLMSNN). In some embodiments, a safety switch caninclude the amino acid sequence of TNF a bound by monoclonal anti-TNF adrugs.

IL-15 and its Receptor in Enhancing CAR T and NK Cell Functions

Recent studies have demonstrated that T cell persistence correlates wellwith CAR T cell therapeutic efficacy. Recent trials demonstrate thatpotent and persistent antitumor activity can be generated by an infusedsmall number of CAR T cells indicative that quality rather than quantityof infused products is more important in contributing to the anti-tumoractivity. Interleukin (IL)-15 is a cytokine that promotes thedevelopment and hemostasis of lymphocytes. Increased levels of IL-15promote T-cell proliferation and enhance T cell effector response. Datafrom recent studies have shown that IL-15 is crucial for the generationand maintenance of memory CD8 T-cells, one of the key factors associatedwith anti-tumor activity. IL-15 binds the IL-15 receptor alpha chain(also called IL-15RA or RA) contributing to IL-15-mediated effects suchas T-cell survival, proliferation and memory T cell generation.

IL-15RA binds the βγ complex in the surface of T cells and IL-15 signalsby binding with this IL-15RA/βγ complex on the cell surface of T cellsand other types of cells.

Transfection of IL-15 alone does not significantly influence T-cellfunction, transfection of IL-15/IL-15RA allows T cells to survive andproliferate autonomously.

The efficacy of administered IL-15 alone may be limited by theavailability of free IL-15RA and its short half-life. Administration ofsoluble IL-15/RA complexes greatly enhanced II-15 half-life andbioavailability in vivo. Therefore, treatment of mice with this complex,but not with IL-15 alone results in robust proliferation and maintenanceof memory CD8 T cells and NK cells. A portion of the extracellularregion of IL-15RA called sushi domain (IL-15sushi) is required for itsbinding of IL-15 (WEI et al., J. Immunol., vol. 167(1), p:277-282,2001). The IL-15/sushi fusion protein is also called IL-15/IL-15sushicontaining the linker is more potent than IL-15 and soluble IL-15RA(IL-15sushi) alone. The combination of IL-15/RA (membrane bound form) orIL-15/sushi (soluble form) can maximize IL-15 activity. The membranebound form, IL-15/RA would not release of free IL-15 by keeping IL-15bound to IL-15RA on the surface of transduced cells.

In the present disclosure, it is unexpected to find that the solubleIL-15/IL-15sushi released from transduced cells are able to promote theexpansion of transduced cells and their neighbor cells, and furtherstimulate them against tumor.

The present disclosure provides an engineered cell having both CAR andIL-15/IL-15sushi or IL-15/RA in a single construct. In some embodiments,the disclosure includes methods to generate higher virus titer and use astronger promoter to drive both CAR and IL-15/RA or IL-15/IL-15sushi.

In some embodiments, the present disclosure provides an engineered cellhaving: (1) a CAR targeting an antigen including, but not limited to,CD4, CD2, CD3, CD7, CD5, CD45, CD20, CD19, CD33, CD123, CS1, and B-cellmature antigen (BCMA); and (2) IL-15; (3) IL-15RA (membrane bound) orsushi (IL-15/IL-15sushi. In further embodiments, CAR comprises chimericantigen receptor, one or more of co-stimulatory endodomains including,but not limited to, CD28, CD2, 4-1BB, 4-1BBL (CD137L), B7-2/CD86,CTLA-4, B7-H1/PD-L1, ICOS, B7-H2, PD-1, B7-H3, PD-L2, B7-H4, CD40Ligand/TNFSF5, DPPIV/CD26, DAP12 and OX40, and intracellular domain ofCD3 zeta chain. In further embodiments, a strong promoter can be, but isnot limited to, SFFV. CARs, IL-15/RA or sushi and inducible suicide gene(“safety switch”), or a combination can be assembled on a vector, suchas a lentiviral vector, adenoviral vector and retroviral vector or aplasmid. The introduction of “safety switch” could significantlyincrease safety profile, and limit on-target or off-tumor toxicities ofCARs.

In one embodiment, the engineered cell includes a CD2 chimeric antigenreceptor polypeptide and IL-15/IL-15sushi (SEQ ID NO.102), andcorresponding polynucleotide (SEQ ID NO. 101). Without wishing to bebound by theory, it is believed that co-expression of IL-15/IL-15sushiwith a CD2 CAR provides long-term durable remissions in patients byincreasing the sensitivity of CAR recognition of target cancer cells orrecruiting innate immune cells to cancer cells.

In one embodiment, the engineered cell includes a CD3 chimeric antigenreceptor polypeptide and IL-15/IL-15sushi (SEQ ID NO.104), andcorresponding polynucleotide (SEQ ID NO. 103). Without wishing to bebound by theory, it is believed that co-expression of IL-15/IL-15sushiwith a CD3 CAR provides long-term durable remissions in patients byincreasing the sensitivity of CAR recognition of target cancer cells orrecruiting innate cells against target cancer cells.

In one embodiment, the engineered cell includes a CD7 chimeric antigenreceptor polypeptide and IL-15/IL-15sushi (SEQ ID NO.106), andcorresponding polynucleotide (SEQ ID NO. 105). In some embodiments, thepresent disclosure provides a method of providing long-term durableremission in a cancer patient by administering a CD7 CAR engineered cellthat co-expresses IL-15/IL-15sushi to a patient in need thereof. Withoutwishing to be bound by theory, it is believed that co-expression ofIL-15/IL-15sushi with a CD7 CAR provides long-term durable remissions inpatients by increasing the sensitivity of CAR recognition of targetcancer cells or recruiting innate immune cells to cancer cells.

In one embodiment, the engineered cell includes a CD5 chimeric antigenreceptor polypeptide and IL-15/IL-15sushi (SEQ ID NO.107), andcorresponding polynucleotide (SEQ ID NO. 108). In some embodiments, thepresent disclosure provides a method of providing long-term durableremission in a cancer patient by administering a CD5 CAR engineered cellthat co-expresses IL-15/IL-15sushi to a patient in need thereof. Withoutwishing to be bound by theory, it is believed that co-expression ofIL-15/IL-15sushi with a CD5 CAR provides long-term durable remissions inpatients by increasing the sensitivity of CAR recognition of targetcancer cells or recruiting innate immune cells to cancer cells.

CAR Targeting CD4+CD25+ Regulatory T Cells

Regulatory T cells (Tregs), also called suppressor T cells, are asub-population of T cells which regulate the immune system and maintaintolerance of self-antigens. Tregs constitute about 1-5% of total CD4+ Tcells in blood with diverse clinical applications in transplantation,allergy, asthma, infectious diseases, graft versus host disease (GVHD),and autoimmunity. The biomarkers for Tregs are CD4, Foxp3 and CD25.Tregs are considered to be derived from Naïve CD4 cells.

In cancers, Tregs play an important role in suppressing tumor immunityand hindering the body's innate ability to control the growth ofcancerous cells.

Tregs expand in patients with cancer and are often enriched in the tumormicroenvironment. Tregs cab infiltrate tumors and limit antitumorimmunity as well. Depletion of Treg can render mice capable of rejectingtumors that normally grow progressively.

Depletion of Tregs using antibodies targeting CD25 results in partialreduction of Tregs but anti-tumor activity is limited. A high-level ofTreg depletion is required for a profound anti-tumor effect. Inaddition, there is a significant issue concerning specificity, as Tregsshare CD25 expression with activated CD4+ and CD8+ lymphocytes as wellas activated NK cells. In general, Tregs are very difficult toeffectively discern from effector T cells and NK cells, making themdifficult to study.

In one embodiment, the engineered cell includes a first chimeric antigenreceptor polypeptide having a CD4 antigen recognition domain and secondchimeric antigen receptor polypeptide having a CD25 antigen recognitiondomain. In one embodiment, this engineered cell includes a polypeptideof SEQ ID NO.92 with a CD45 leader sequence and correspondingpolynucleotide of SEQ ID NO. 91.

In one embodiment, the engineered cell includes a first chimeric antigenreceptor polypeptide having a CD4 antigen recognition domain and secondchimeric antigen receptor polypeptide having a CD25 antigen recognitiondomain. In one embodiment, this engineered cell includes a polypeptideof SEQ ID NO.94 with a CD8a leader sequence and correspondingpolynucleotide of SEQ ID NO. 93.

Specific Embodiments for T-Regulatory Cells

T lymphocytes (T cells) are a subtype of white blood cells that play akey role in cell-mediated immunity. T cells are divided into CD4 and CD8cells. Natural killer cells (NK cells) are a type of cytotoxic cellscritical to the innate immunity.

T-regulatory cells (Tregs) are a type of CD4+ cells mediating immunetolerance and suppression and are distinguished from helper T cells.Tregs express CD4, CD25 and Foxp3 (CD4CD25+ regulatory T cells).

Tregs are enriched in the tumor microenvironment and considered to beimportant for hindering antitumor immune responses and promoting cancercell tolerance. Increased numbers of infiltrating Tregs in tumors havebeen associated poor survival in a variety of cancers includinghematologic malignancies and solid tumors.

Tregs appear to be preferentially trafficked to the tumormicroenvironment and play a critical role of immunosuppression (Ethan M.Shevach et al, Annual Review of Immunology, Vol. 18: 423-449, 2000).

A number of different methods are employed to delete Tregs for cancertreatments by targeting CD25, resulting in a partial reduction of Tregs.However, this could be problematic as: (1) CD25 is also expressed inactivated CD4, CD8 and NK cells. CD25 expression can be seen inactivated B cells, macrophages, osteoblasts, some thymocytes, somemyeloid precursors and some oligodendocytes. (2) a very high-level ofTreg depletion is required for efficacy (Xingrui Li et al, Eur. J.Immunol. 2010. 40: 3325-3335).

The CAR design is to redirect patient or donor immune cells against aspecific “target” antigen in a major-histocompatibility complex(MHC)-independent manner. The CAR protein construct usually includes anumber of modular components or regions integral to function. The modulefor “target” recognition is the extracellular single-chain variablefragment (scFv). This component is derived from a monoclonal antibodywith specific direction against a carefully selected target antigen. Ahinge region of appropriate length tandem to the scFv conveys mobilityof the scFv region to allow for optimal binding to the target protein.The transmembrane domain region serves as a conduit between theextracellular binding regions and co-activation domains' such as CD28and/or 4-1BB. The final module includes the CD3 zeta intracellularsignaling domain.

The present disclosure provides a method for a novel Treg CAR (alsocalled CD4zetaCD25CAR or C4-25z CAR) construct targeting a cellco-expressing CD4 and CD25. The Treg CAR depletes Tregs specificallywhile sparing most of cells that do not co-express CD4 and CD25.

In some embodiments, T cell activation could be achieved uponsimultaneous engagement of two scFv molecules against CD4 and CD25 in aTreg CAR. In a further embodiment, both T cell activation andco-stimulation signals are provided using two distinct/separate chimericantigen receptor polypeptides.

In some embodiments, a TregCAR includes (1) a first chimeric antigenreceptor polypeptide unit comprising a first signal peptide, a firstantigen recognition domain, a first hinge region, a first transmembranedomain, and an intracellular signaling domain; and (2) a second chimericantigen receptor polypeptide unit comprising a second signal peptide, asecond antigen recognition domain, a second hinge region, a secondtransmembrane domain, and a co-stimulatory domain (s). Both the firstchimeric antigen receptor polypeptide unit and the second chimericengineered polypeptide unit are expressed on a single polypeptidemolecule, and wherein an amino acid sequence comprising a highefficiency cleavage site is disposed between the first chimeric antigenreceptor polypeptide unit and the second chimeric antigen receptorpolypeptide unit.

In some embodiments, the Treg CAR potentiates the lysis activity of acell co-expressing CD4 and CD25 while minimizing a cell bearing only CD4or CD25 antigen.

In some embodiments, the nucleotide sequence of the first chimericantigen receptor polypeptide unit is different from the second chimericengineered polypeptide unit in order to avoid a homologous recombination

In some embodiments, the high efficiency cleavage site in Treg CAR isP2A.

In some embodiments, the target of the first antigen recognition domainis either CD4 or CD25 and the target of the second antigen recognitiondomain is either CD4 or CD25; wherein the first antigen recognitiondomain is different than the second antigen recognition domain.

In one embodiment, the antigen recognition domain includes the bindingportion or variable region of a monoclonal or polyclonal antibodydirected against (selective for) the target.

In a further embodiment, the target antigen is CD4 or CD25.

In some embodiments, the T or NK cell comprising Treg CARs targetingdifferent or same antigens.

In some embodiments, the T or NK cell comprises Treg CARs targetingTregs expressing CD4 and CD25 while sparing most of cells, which do notco-express CD4 and CD25.

In some embodiments, the T or NK cell comprises Treg CARs depletingTegs.

In some embodiments, the present disclosure provides a method ofgeneration of Treg CAR useful for treating or preventing aCD4+CD25+Foxp3+ T regulatory cell (Treg) related disease in a subject isprovided. In a further embodiment, the diseases treated with Treg CARinclude, but not limiting to, cancers.

In some embodiments, the present disclosure provides a method ofgeneration of Treg CAR useful for treating or preventing aCD4+CD25+Foxp3+ T regulatory cell (Treg) related Cancers including, butnot limited, hepatocellular carcinoma, fibrolamellar carcinoma,hepatoblastoma, undifferentiated embryonal sarcoma and mesenchymalhamartoma of liver, lung-squamous cell carcinoma, testicularnonseminomatous germ cell tumors, liposarcoma, ovarian and extragonadalyolk sac tumors, ovarian choriocarcinoma, teratomas, ovarian clear cellcarcinoma, placental site trophoblastic tumor, lymphoma and leukemia.

In some embodiments, the present disclosure provides a method ofgeneration of Treg CAR useful for inhibiting the growth of a tumor in asubject is provided.

In some embodiments, the Treg CAR is administrated in combination withany chemotherapy agents currently being developed or available in themarket. In some embodiments, the Treg CAR is administrated as a firstline treatment for diseases including, but not limited to, hematologicmalignancies and cancers.

In some embodiments, the cells expressing a Treg CAR areco-administrated with immunomodulatory drugs, such as, but not limitedto, CTLA-4 and PD-1/PD-L1 blockades, or cytokines, such as IL-2, IL-15or IL-15/IL-15sushi or IL-15/RA, and IL-12 or inhibitors of colonystimulating factor-1 receptor (CSF1R) for better therapeutic outcomes.

In some embodiments, the cells expressing a Treg CAR areco-administrated with other immunomodulatory drugs or CAR-expressingcells to provide synergistic effects in a subject.

In a specific embodiment, the cells expressing a Treg CAR can be T cellsor NK cells, administrated to a mammal, e.g. human.

In some embodiments, the Treg CAR is used in immunotherapy in thetreatment of cancers. The cancers may be selected from the groupconsisting of lung cancer, melanoma, breast cancer, prostate cancer,colon cancer, renal cell carcinoma, ovarian cancer, cervical cancer,head or neck cancer, stomach cancer, liver cancer, neuroblastoma,rhabdomyosarcoma, leukemia and lymphoma. The compositions and methodsdescribed in the present disclosure may be utilized in conjunction withother types of therapy for cancer, such as chemotherapy, surgery,radiation, gene therapy, and so forth.

To achieve enhanced safety profile or therapeutic index, the Treg CAR ofthe present disclosure may be constructed as a transient RNA-modified“biodegradable” version or derivatives, or a combination thereof. TheRNA-modified CARs of the present disclosure may be electroporated into Tcells or NK cells. The expression of the Treg CAR may be graduallydiminished over few days.

A method for treating cancers using Treg CAR in a subject is embodied inthe present disclosure. The method comprises:

-   -   (1) Obtaining T/NK cells from a subject or donor(s).    -   (2) Culturing the lymphocytes/T cells or NK cells.    -   (3) Introducing an Treg CAR construct into the cultured T cells        or NK cells.    -   (4) Expanding Treg CAR T cells or NK cells    -   (5) Treating the subject by administering a therapeutically        effective amount thereto.

The ex vivo expansion of tumor-infiltrating lymphocytes (TILs) aresuccessfully used in the current adoptive cell therapy. In oneembodiment, TILs are harvested and successfully expanded ex vivo.

In some embodiments, TILs can be obtained from a tumor tissue sample andexpanding the number of TILs. Treg CAR T or NK cells were used toco-culture with TILs to deplete Treg population to enhance TIL responsesto cancers, which is valuable to the disease therapies.

In some embodiments, CD4CAR bear a set of CAR body including an antigenrecognition domain, a hinge region, a co-stimulatory domain (s) and anintracellular domain (CD3 zeta chain). In a further embodiment, CD4CARdepletes Tregs, and then enhances T-cell 10 responses to cancer cellsand improves therapeutic outcomes of anti-tumor activity.

In some embodiments, CD25CAR bear a set of CAR body including an antigenrecognition domain, a hinge region, a co-stimulatory domain (s) and anintracellular domain (CD3 zeta chain). In a further embodiment, CD25CARdepletes Tregs, and then enhances T-cell responses to cancer cells andimproves therapeutic outcomes of anti-tumor activity.

The present disclosure may be better understood with reference to theexamples, set forth below. The following examples are put forth so as toprovide those of ordinary skill in the art with a complete disclosureand description of how the compounds, compositions, articles, devicesand/or methods claimed herein are made and evaluated, and are intendedto be purely exemplary and are not intended to limit the disclosureAdministration of any of the engineered cells described herein may besupplemented with the co-administration of a CAR enhancing agent.Examples of CAR enhancing agents include immunomodulatory drugs thatenhance CAR activities, such as, but not limited to agents that targetimmune-checkpoint pathways, inhibitors of colony stimulating factor-1receptor (CSFIR) for better therapeutic outcomes. Agents that targetimmune-checkpoint pathways include small molecules, proteins, orantibodies that bind inhibitory immune receptors CTLA-4, PD-1, andPD-LI, and result in CTLA-4 and PD-1/PD-L1 blockades. As used herein,enhancing agent includes enhancer as described above.

As used herein, “patient” includes mammals. The mammal referred toherein can be any mammal. As used herein, the term “mammal” refers toany mammal, including, but not limited to, mammals of the orderRodentia, such as mice and hamsters, and mammals of the orderLogomorpha, such as rabbits. The mammals may be from the orderCarnivora, including Felines (cats) and Canines (dogs). The mammals maybe from the order Artiodactyla, including Bovines (cows) and Swines(pigs) or of the order Perssodactyla, including Equines (horses). Themammals may be of the order Primates, Ceboids, or Simoids (monkeys) orof the order Anthropoids (humans and apes). Preferably, the mammal is ahuman. A patient includes subject.

In certain embodiments, the patient is a human 0 to 6 months old, 6 to12 months old, 1 to 5 years old, 5 to 10 years old, 5 to 12 years old,10 to 15 years old, 15 to 20 years old, 13 to 19 years old, 20 to 25years old, 25 to 30 years old, 20 to 65 years old, 30 to 35 years old,35 to 40 years old, 40 to 45 years old, 45 to 50 years old, 50 to 55years old, 55 to 60 years old, 60 to 65 years old, 65 to 70 years old,70 to 75 years old, 75 to 80 years old, 80 to 85 years old, 85 to 90years old, 90 to 95 years old or 95 to 100 years old.

The terms “effective amount” and “therapeutically effective amount” ofan engineered cell as used herein mean a sufficient amount of theengineered cell to provide the desired therapeutic or physiological oreffect or outcome. Such, an effect or outcome includes reduction oramelioration of the symptoms of cellular disease. Undesirable effects,e.g. side effects, are sometimes manifested along with the desiredtherapeutic effect; hence, a practitioner balances the potentialbenefits against the potential risks in determining what an appropriate“effective amount” is. The exact amount required will vary from patientto patient, depending on the species, age and general condition of thepatient, mode of administration and the like. Thus, it may not bepossible to specify an exact “effective amount”. However, an appropriate“effective amount” in any individual case may be determined by one ofordinary skill in the art using only routine experimentation. Generally,the engineered cell or engineered cells is/are given in an amount andunder conditions sufficient to reduce proliferation of target cells.

Following administration of the delivery system for treating,inhibiting, or preventing a cancer, the efficacy of the therapeuticengineered cell can be assessed in various ways well known to theskilled practitioner. For instance, one of ordinary skill in the artwill understand that a therapeutic engineered cell delivered inconjunction with the chemo-adjuvant is efficacious in treating orinhibiting a cancer in a patient by observing that the therapeuticengineered cell reduces the cancer cell load or prevents a furtherincrease in cancer cell load. Cancer cell loads can be measured bymethods that are known in the art, for example, using polymerase chainreaction assays to detect the presence of certain cancer cell nucleicacids or identification of certain cancer cell markers in the bloodusing, for example, an antibody assay to detect the presence of themarkers in a sample (e.g., but not limited to, blood) from a subject orpatient, or by measuring the level of circulating cancer cell antibodylevels in the patient.

Throughout this specification, quantities are defined by ranges, and bylower and upper boundaries of ranges. Each lower boundary can becombined with each upper boundary to define a range. The lower and upperboundaries should each be taken as a separate element.

Reference throughout this specification to “one embodiment,” “anembodiment,” “one example,” or “an example” means that a particularfeature, structure or characteristic described in connection with theembodiment or example is included in at least one embodiment of thepresent embodiments. Thus, appearances of the phrases “in oneembodiment,” “in an embodiment,” “one example,” or “an example” invarious places throughout this specification are not necessarily allreferring to the same embodiment or example. Furthermore, the particularfeatures, structures or characteristics may be combined in any suitablecombinations and/or sub-combinations in one or more embodiments orexamples. In addition, it is appreciated that the figures providedherewith are for explanation purposes to persons ordinarily skilled inthe art and that the drawings are not necessarily drawn to scale.

As used herein, the terms “comprises,” “comprising,” “includes,”“including.” “has,” “having,” or any other variation thereof, areintended to cover a non-exclusive inclusion. For example, a process,article, or apparatus that comprises a list of elements is notnecessarily limited to only those elements but may include otherelements not expressly listed or inherent to such process, article, orapparatus.

Further, unless expressly stated to the contrary, “or” refers to aninclusive “or” and not to an exclusive “of”. For example, a condition Aor B is satisfied by any one of the following: A is true (or present)and B is false (or not present), A is false (or not present) and B istrue (or present), and both A and B are true (or present).

Additionally, any examples or illustrations given herein are not to beregarded in any way as restrictions on, limits to, or expressdefinitions of any term or terms with which they are utilized. Instead,these examples or illustrations are to be regarded as being describedwith respect to one particular embodiment and as being illustrativeonly. Those of ordinary skill in the art will appreciate that any termor terms with which these examples or illustrations are utilized willencompass other embodiments which may or may not be given therewith orelsewhere in the specification and all such embodiments are intended tobe included within the scope of that term or terms. Language designatingsuch nonlimiting examples and illustrations includes, but is not limitedto: “for example,” “for instance,” “e.g.,” and “in one embodiment.”

In this specification, groups of various parameters containing multiplemembers are described. Within a group of parameters, each member may becombined with any one or more of the other members to make additionalsub-groups. For example, if the members of a group are a, b, c, d, ande, additional sub-groups specifically contemplated include any one, two,three, or four of the members, e.g., a and c; a, d, and e; b, c, d, ande; etc.

As used herein, a XXXX antigen recognition domain is a polypeptide thatis selective for XXXX. “XXXX” denotes the target as discussed herein andabove. For example, a CD38 antigen recognition domain is a polypeptidethat is specific for CD38.

As used herein, CDXCAR refers to a chimeric antigen receptor having aCDX antigen recognition domain.

As used herein, a CAR engineered cell is an engineered cell as describedherein that includes a chimeric antigen receptor polypeptide. By way ofexample, a CD45 engineered cell is an engineered cell having a CD45chimeric antigen receptor polypeptide as disclosed herein.

As used herein, a compound CAR (cCAR) engineered cell is an engineeredcell as described herein that includes at least two distinct chimericantigen receptor polypeptides. By way of example, a CD19CD22 compoundCAR engineered cell is an engineered cell as described herein thatincludes a first chimeric antigen receptor polypeptide having a CD19antigen recognition domain, and a second chimeric antigen receptorpolypeptide having a CD22 antigen recognition domain.

The present disclosure may be better understood with reference to theexamples, set forth below. The following examples are put forth so as toprovide those of ordinary skill in the art with a complete disclosureand description of how the compounds, compositions, articles, devicesand/or methods claimed herein are made and evaluated, and are intendedto be purely exemplary and are not intended to limit the disclosure.

EXAMPLES Engineered cCAR Targets Cells Expressing CD33 or CD123 or BothGeneration of Compound CAR (cCAR)

The construction of the CD33CD123 cCAR follows the schematic in FIGS. 1and 2A. It includes SFFV (spleen focus-forming virus) promoter thatdrives the expression of the functional compound CAR (cCAR) bearing twodifferent units of CARs. The antigen receptor head, a scFv (single-chainvariable fragment) nucleotide sequence of the anti-CD33 and anti-CD123.A P2A peptide derived from picornavirus is utilized due to the highlyefficient mechanism of its self-cleaving dynamics for bicistronicgenetic constructs. The self-cleaving P2A peptide serves to link the twoindependent units of CARs, CD33CAR, and CD123CAR together duringexpression. The advantages of this approach over an internal ribosomalentry site (IRES), which is commonly used in the literature, include itssmall size and high cleavage efficiency between two unit proteinsupstream and downstream of the 2A peptide. In addition, the use ofself-cleaving P2A peptide can avoid a problem of differences inexpression levels between gene before and after IRES when IRES isapplied.

The modular unit, CD33CAR includes the CD33 scFv domain, a CD8a hingeregion, a CD8a transmembrane domain, 4-BB co-stimulatory domain and anintracellular domain of CD3 zeta chain. The second modular CAR, CD123CARbears the same hinge, transmembrane and intracellular signaling domainsas CD33CAR but different scFv, and co-stimulatory domains. The CD33 CARrecognizes its corresponding antigen and the CD123 CAR binds to itscorresponding antigen. The hinge region was designed such that sequenceswhere disulfide interactions are avoided. Different co-stimulatorydomains, 4-BB and CD28 were used. The CD33CD123 compound CAR wassubcloned into a lentiviral plasmid.

Generation of a High-Efficiency Compound CAR (cCAR)

Compound CAR lentivirus was generated by transfection of HEK-293 FTcells with Lipofectamine 2000 according to manufacturer's directions,except with 2× the vector DNA due to a large size of insert, in order toincrease titer as shown in FIG. 2 . After about 12-16 hours incubation,media containing Lipofectamine was removed and replaced with DMEMcontaining 10% FBS, 20 mM HEPES, 1 mM sodium pyruvate and 1 mM sodiumbutyrate. After about 24 hours, the supernatant was harvested andrefrigerated, and replaced with fresh media. After about another 24hours, this was collected and combined with the previous supernatant,and filtered through a 0.45 μM filter disc. Supernatant was split intoaliquots, flash frozen with liquid nitrogen and stored at −80° C.HEK-293 FT cells were harvested, stored frozen, and lysed for subsequentelectrophoresis and Western blotting (FIG. 2B).

PB (peripheral blood) or CB (human umbilical cord blood) buffy coatcells were activated 2 days with anti-CD3 antibody and IL-2. cCARlentiviral supernatant was spinoculated onto retronectin-coatedmultiwell plates. Activated T cells were transduced in multiple wellswith lentiviral supernatant at a low concentration of about 0.3×10⁶cells/mL to increase transduction efficiency (FIG. 3 ).

Following the first overnight transduction, cells were added directly toa second virus-coated plate for a second transduction without washing,unless the cells did not look healthy. Following the second overnighttransduction, cells were washed, combined and incubated in tissueculture treated plates. CAR T cells were allowed to expand for up toabout 5 days prior to co-culture killing assays. After about 3 days ofincubation, cells were incubated with goat anti-mouse F(Ab′)2 or goatIgG (isotype) antibodies conjugated with biotin, washed and followed byincubation with streptavidin-PE and conjugated anti-human CD3. Afterwashing and suspension in 2% formalin, cells were then analyzed by flowcytometry to determine percent transduction efficiency.

Characterization of the CD33CD123 cCAR

Transfected CD33CD123 cCAR HEK293T cells were subjected to Western blotanalysis in order to confirm the compound construct. Immunoblot with ananti-CD3ζ monoclonal antibody showed bands of predicted size for thecompound CAR CD3ζ fusion protein (FIG. 2B). Importantly, two distinctbands of similar intensity were observed on the blot signaling thesuccessful high cleavage action of the P2A peptide as expected. No CD3ζexpression was seen for the GFP control vector as expected. The surfaceexpression of scFv was also tested on HEK 293 cells (FIG. 2C) andprimary T cells (FIG. 2C).

The compound CD33CD23CAR lentivirus was tested for transductionefficiency in the HEK293 cell line and analyzed by flow cytometry(Beckman Coulter) (FIG. 2C). Flow cytometry showed that about 67% of HEKcells expressed CD33CD123 CARs. Human peripheral blood (PB) is oftenused for autologous T cell therapy. Human PB buffy coat cells wereactivated with anti-CD3 antibody and IL-2, and transduced with eitherCD4CAR or control (GFP) lentiviruses. After transduction, flow cytometryanalysis showed that about 22% of T-cells expressed the CD33CD123CAR(FIG. 2C).

CD33CD123 cCAR T-Cells Derived from Umbilical Cord Blood (UCB) andPeripheral Blood (PB) Specifically Kill CD33-Expressing Tumor Cells

CD33CD123 cCAR T cells or GFP T cells (control) were incubated withtarget cells at ratios ranging from 0.5:1 from 50:1, preferably, atabout 2:1, 5:1, 10:1, 20:1, 50:1, at about 100,000, 200,000, 500,000,about 1 million, or 2 million effector cells to about 50,000, 100,000,200,000 target cells, respectively) in about 1-2 mL T cell culturemedia, without IL-2 for about 24h. Target cells were leukemic cell linesand leukemia cells from a patient with leukemia. After about 24 hours ofco-culture, cells were stained with mouse anti-human CD33, CD123, CD34and CD3 antibodies.

CD33CD123 cCAR T cells expressing the CD33CAR and CD123 CAR weregenerated and tested for anti-leukemic functions using the HL60 andKG-1a cell lines. The HL60 cell line is a promyelocytic leukemia cellline highly enriched for CD33. About 100% of its cell population isCD33+ with a small subset (<10%) of it being dim CD123+. In culture,this cell line was tested to determine the effectiveness of theCD33CD123 CAR with an emphasis on targeting CD33-expressing leukemiccells. Additionally, due to the strong expression of CD33 in HL60, it isCD33CD123 cCAR action may be profound. Indeed, during 24h co-cultureconditions with various ratios of effector to target cells, theCD33CD123 cCAR exhibited significant leukemic cell killing properties(FIG. 4 ). CB-derived CD33CD123 CAR T-cells were first tested for theirability to kill HL60 cells. At about 24h incubation and loweffector:target (E:T) ratios ranging from about 0.5:1 to 50:1,preferably, 1:1 to about 5:1, more preferably about 2:1 to 4:1,CD33CD123 CAR cells eliminated about 55% of the CD33 expressing HL60cells when compared to GFP control. At a ratio of about 5:1, the killingaction rose to about 82%.

CD33CD123 CAR derived from peripheral blood mononuclear cells (PBMCs)were co-cultured with the myelogenous leukemia cell line KG1a, whichalso expresses about 100% CD33 at moderate levels compared to HL60 and50-80% CD123. KG1a is, therefore, a relatively dual target cellpopulation that is double positive for the antigens targeted by theCD33CD123 CAR. At about 24 hours of incubation and low effector:target(E:T) ratios ranging from about 0.5:1 to 50:1 were used. While at a lowE:T ratio of about 2:1, the CD33CD123 CAR exhibited modest anti-leukemicactivity about 26%, an increase in E:T ratio to 10:1 resulted in akilling of KG1a of about 62% compared to GFP control (FIG. 5 ),signaling that the intensity of the CD33 marker may be an indicator forthe efficacy of killing with HL60 presenting strongly and harnessingmore CAR action than KG1a. These experiments provide evidence for thefunction of the whole CD33CD123 CAR against its relevant antigenpresenting cell populations.

Additional compound CAR, CD33CD123-BB cCAR has been generated (data notshown). This compound CAR comprises two independent units of CARs, CD33and CD123. The first CAR comprises scFv binding to CD33 and the secondCAR bears a different scFv recognizing CD123. Both CARs contain the samehinge region, transmembrane, co-stimulatory and intracellular domains.CD33CD123-BB cCAR lentiviruses were produced and their killing abilitywas tested in KG-1a cells. As shown in FIG. 5 , there was substantialkilling at a ratio of about 10:1 but it is less potent than that ofCD33CD123 cCAR.

CD33CD123 cCAR Possesses Activity Against Patient Samples ExpressingCD33 and/or CD123

In addition to cell line experiments, studies were also conducted onpatient samples in order to test the function of each individual CARunit. An aggressive acute myeloid leukemia (AML), AML-9 was used fortesting efficacy of the CD33CD123 cCAR. Due to the heterogeneity of thepatient cell population, which includes multiple cell types in the AML-9sample, leukemic blasts were gated with CD34 and CD33, as they werepositive for these two markers. The depletion of this CD33+CD34+population of leukemic cells was observed to be 48% over the GFP controlat a ratio of CAR T cell:target cell (FIG. 6 ).

Leukemic cells that were CD123 positive and CD33 negative were alsotested. For this purpose, human B cell acute lympoblastic leukemia(B-ALL) sample, Sp-BM-B6 was chosen. All leukemic blasts in this samplewere CD34+CD33−, and more than about 50% positive for CD123. Depletionof the CD34+ leukemic cell population by CD33CD123 cCAR T cells wasabout 86% as compared to that of the GFP control (FIG. 7 ). Based on thecell line and human sample studies, our data strongly suggest that thecompound CD33CD123 CAR is able to target leukemic cells expressing CD33or CD123 or both.

CD33CD123 cCAR NK Cells Targeting Leukemia Cells Expressing CD33 or CD23or Both

Natural killer (NK) cells are CD56+CD3− and can efficiently killinfected and tumor cells like CD8+ T cells. Unlike CD8+ T cells, NKcells launch cytotoxicity against tumors without the requirement ofactivation to kill cells. NK cells are safer effector cells, as they mayavoid the potentially lethal complications of cytokine storms. However,the use of either CD33 or CD123 or both CAR NK cells in killingleukemias is entirely unexplored.

Production of CD33CD123 cCAR NK Cells

NK-92 cells were transduced with CD33CD123 CAR lentiviral supernatant intwo consecutive overnight transductions with a change of retronectin-and virus-coated plates in between. The transduced cells were expandedfor 3 or 4 days and then analyzed by flow cytometry for CAR expression.Cells were harvested and incubated with goat anti-mouse F(Ab′)2 at about1:250 for about 30 minutes. Cells were washed, suspended and stainedwith streptavidin-PE for about 30 minutes. Cells were washed andsuspended in 2% formalin, and analyzed by flow cytometry. NK-92 cellsexpressing CD33CD123 cCAR were then labeled as above and sorted onFACSAria, with the top 0.2% of F(Ab′)2-expressing cells collected andcultured. Subsequent labeling of sorted, expanded cells showed about 89%of NK-92 cell positive for anti-mouse F(Ab′)2 (FIG. 8 ).

CD33CD123 cCAR NK Cells Efficiently Lyse or Eliminate Leukemic Cells

First, we tested the function of CD33CD123 cCAR NK-92 cells by assessingtheir ability to kill a HL-60 cancerous cell line in co-culture.Virtually all HL-60 cells highly express CD33 but CD123 expression inthis cell line is only less than 10% (weak). Therefore, it is likelythat the killing ability of CD33CD123cCAR is dependent on the abilityfor cCAR to properly targeting CD33.

CD33CD123 cCAR NK-92 cells were co-cultured with the HL-60 cells forabout 24 hours in NK cell media without IL-2. After the incubation, theCD33CD123 cCAR NK-92 cells were labeled and compared to a control ofnon-CAR, GFP NK-92 cells. Dramatic killing of HL-60 cells by CD33CD123cCAR NK-92 cells was observed as compared to the control, GFP NK-92cells. Moreover, the killing ability of CD33CD123 cCAR NK-92 cells wasdose-dependent, with a about 10 to 1 ratio of about 100% compared to thecontrol (FIGS. 9 and 11 ).

A second co-culture experiment using the myeloid leukemia cell line wasperformed using KG1a, which expresses CD33 in all cells but at amoderate level compared to that of HL-60. The CD123 antigen is expressedin about 50-80% of KG1a cells. The experimental design was similar tothe first experiment of the HL-60 killing assay described above, withthe same incubation time, effector:cancer cell ratios and GFP NK-92 cellcontrols. Results show a remarkable killing of KG1a cells by CD33CD123cCAR NK-92 cells in a dose-dependent manner as compared to the GFP NK-92cell control. At a ratio of effector: target of 10:1, killing of KG1acells by CD33CD123 cCAR NK-92 cells was about 85% as compared to that ofGFP control (FIGS. 10 and 11 ).

Analysis of KG1a cells showed two different populations, CD33+CD123− andCD33+CD123−. FIG. 11 showed a dose dependent increase in cell killingseen in both populations. Surprisingly, the double positive populationshowed a higher efficient killing for each increased ratio, suggesting apossible synergistic effect of two modular CARs of CD33 and CD123 (FIG.12 ).

We also generated engineered CD33CD123 CAR T cells received not onlycostimulation through the CD28 but also co-express the 4-1BB ligand(4-1BBL or CD137L) in a single construct, which provide the bettertherapeutic efficacy (FIG. 13A). T-cells derived from peripheral bloodfrom healthy donors were transduced with the CD33CD123-4-1BBL-2Gconstruct in 6-well plates incubated with 2 ml of virus supernatant. CARexpression was assayed with F(ab)′ labeling for surface expression ofthe CAR protein and subsequently underwent FACS analysis. Transducedcells were compared to control T-cells labeled at the same time.Expression was determined and transduced population encircled on plot 1day after the end of transduction period. The surface CD33CD123-41BBL-2GCAR expression on T cells was approximately 60% (FIG. 13B). CD33CD123CAR improves functional activates when 4-1BBL was included in theconstruct.

An enhancer, IL-15/IL-15sushi was also included in CD33CD123 CARconstruct as an alternative approach. Both compound CAR, CD33CD123 andIL-15/IL-15sushi were in a single construct (FIG. 14 ). IL-15/IL-15sushiis able to promote the expansion of CAR T/NK cells, and infiltrate ofinnate immune cells to the tumor site, which could result in bettertumor destruction

Engineered cCAR Targets Cells Expressing: 1) CD19 or CD20 or Both; 2)CD19 or CD20 or Both; 3) CD19 or CD138 or Both

Generation of CD19CD20, CD19CD22, and CD19CD138 cCARs

The three cCARs have been generated (FIG. 15 ) using the similarstrategy to that of the CD33CD123 cCAR described above.

Generation of the Second Generation Compound CARs (CD19CD20 andCD19CD22)

The construction of the compound CAR (cCAR) follows the schematic inFIG. 16A. It comprises of SFFV (spleen focus-forming virus) promoterthat drives the expression of the functional cCAR bearing two differentunits of CARs. The first CAR is the complete L8-CD19-2G CAR (using humanCD8a leader sequence, called L-8), linked to the complete second CAR(either CD20-2G or CD22-2G) by a high efficient P2A self-cleavingpeptide, derived from picornavirus. The entire sequence is in frame asto result in initially one large fusion protein which is cleaved in halfprior to cell surface expression. This method ensures equal expressionlevels of both CARs. The cCAR DNA molecules were subsequently sub-clonedinto the same lentiviral plasmid as above.

Transduced T Cells Efficiently Express cCARsLentiviral vector supernatant was generated from HEK293T cellstransfected with either CD19CD20-2G or CD19CD22-2G vector construct orcontrol vector. After collection of lentiviral supernatant wascollected, cells were harvested, lysed, and electrophoresed prior toWestern blot transfer. Incubation of blot membrane with anti-humanCD3zeta antibody resulted in two distinct bands representing each CARunit after cleavage; the CD19CAR is slightly larger than the CD20 orCD22 CAR units (FIG. 18B). Next, peripheral blood mononuclear buffy coatcells were activated for three days and transduced with concentratedCD19CD20-2G, CD19CD22-2G or control vector lentiviral supernatant onnon-tissue culture plates coated with retronectin. The transductionprocedure was repeated 24 hours after the first transduction. CARexpression on the T-cell surface was demonstrated three days aftertransduction by staining transduced T cells with goat anti-mouse F(Ab′)2antibody and mouse anti-human CD3.

FIG. 16 shows that 26.9% of cells transduced with concentratedL8-CD19CD20-2G lentiviral supernatant and 35.6% of T cells transducedwith concentrated CD19CD22-2G lentiviral supernatant were positive forboth F(Ab′)2 and CD3 as determined by flow cytometry, when compared tothe control transduction.

Transduced T Cells Express CD19CD22-2G at Different Levels Based onLeader Sequences

We then determined the leader sequence that would result in the highestlevel of cell surface expression of cCAR, three constructs were madethat incorporated leader sequences for human CD8a (L8), CD45 (L45), andcolony stimulating factor (CSF) (FIG. 17 ). Following transduction ofhuman peripheral blood T cells with lentiviral supernatant generatedfrom each of these vectors, transduction efficiency for the T cells wasdetermined using F(Ab′)2 antibody as above. The L8 leader sequence againled to the highest transduction efficiency (43.8%), followed by L45(9.8%) and CSF (1.3%). (FIG. 17 ). This shows that the optimal design ofa compound CAR, like a single CAR, depends in part on the leadersequence for surface CAR expression.

Concentration of Lentiviral Supernatant can Lead to Higher TransductionEfficiency for cCARs

To improve CAR efficiency in transduced T cells, lentiviral supernatantfor CD19CD20-2G and CD19CD22-2G was centrifuged at 3,880×g for 24 hours.The resulting viral pellets were suspended in media at one third theiroriginal volume, making them 3× concentrated. This concentrate was usedto transduce activated T cells in the same volume as non-concentratedvirus. FIG. 10 a shows that CAR efficiency for T cells transduced with3× concentrated CD19CD22-2G lentiviral supernatant nearly tripled, whileCAR efficiency for T cells transduced with 2.5× concentrated CD19CD20-2Glentiviral supernatant increased nearly 10-fold (FIG. 18 ). Thisillustrates the importance of concentrating lentiviral vector for thelonger cCAR constructs.

cCAR CAR T Cells Specifically Target CD19-Expressing Tumor Cell Lines

T cell co-culture killing assays were performed to determine the abilityof L8-CD19CD22-2G and L8-CD19CD20-2G CAR T cells to effectively lyse theCD19+ cell lines, SP53 and JeKo-1 (both mantle cell lymphoma lines).Briefly, each target cell line was pre-labeled with CMTMR membrane dye,and then co-cultured with either vector control, L8-CD19CD22-2G orL8-CD19CD20-2G CAR T cells at ratios of 2:1 and 5:1 effector:targetcells (200,000 or 500,000 effector cells to 100,000 target cells, in 1mL T cell media without serum or IL-2). After overnight incubation,cells were labeled with anti-human CD3-PerCp and CD19-APC for 30minutes, washed, and suspended in 2% formalin for analysis by flowcytometry. The L8-CD19CD22-2G CAR T cells demonstrated robust lysis oftumor cells (FIG. 19 ), lysing 53.4% and 93% of the SP53 cells at 2:1and 5:1 ratios, respectively. At the same ratios, the L8-CD19CD22-2G CART cells were able to lyse 69% and 97.3% of the JeKo-1 cells (FIG. 20 ).

cCAR CAR T Cells Eliminate CD19+ Cells from AML and B-ALL PatientSamples

Studies were again conducted using patient samples. Buffy coat fractionsof these primary cells were pre-labeled with CMTMR and co-cultured witheither vector control, or L8-CD19CD22-2G CAR T cells in the same manneras the tumor cell lines. L8-CD19CD22-2G CAR T cells lysed 54.3% and 77%of the AML patient cells with aberrant expression of CD19 at 2:1 and 5:1ratios, respectively, in an overnight co-culture, and lysed 84.3% of theB-ALL tumor cells at a 1:1 ratio in a four day co-culture with 2.5% FBSand IL-2 added to the media (FIGS. 21, 22A). As these AML patient cellsonly comprised 65% blasts and 75% of them expressed CD19, it was likelythat L8-CD19CD22-2G CAR T cells were able to eliminate the entire CD19positive blast population.

cCAR CAR T Cells Lyse K562 Cells Expressing CD22.An artificial K562 expressing CD22 cell line (K562xp22) via transductioninto wild-type K562 cells was generated. Subsequently, we tested theanti-tumor properties of the CD19CD22 cCAR to target the minor CD22⁺population of the K562 cells. A co-culture experiment at 1:1 ratio(effective: target) show a modest significant cytotoxic effect on K562expressing CD22 population compared to the control. Cytotoxicity resultsremain consistent with other numbers reported for anti-tumor activityagainst artificial antigen presenting cell lines (FIG. 22B).

Engineered cCAR Targets Cells Expressing BCMA and CS1

Generation of cCAR Including BCMA CS1 cCAR and BCMA CD19 cCAR forTreatment of Multiple Myeloma or Autoimmune Disorders

Pre-clinical studies have been developed for cCARs to target surfaceantigens including CD38, CS1, CD138, B cell maturation antigen (BCMA)and CD38. CD19 CAR has also demonstrated some efficacy for the treatmentof multiple myeloma in a phase I clinical trial. However, given that theheterogeneity of surface antigen expression commonly occurs in malignantplasma cells, it is unlikely that a single target is sufficient toeliminate this disease. BCMA CS1 cCAR, BCMA CD19 cCAR, BCMA CD38 cCARand BCMA CD138 cCAR were generated and the experimental design wassimilar to that of CD33CD123 cCAR as described above.

Generation of cCAR Including BCMA CS1 cCAR (BC1cCAR) for Treatment ofMultiple Myeloma or Autoimmune Disorders

Generation and Characterization of BCMA-CS1 cCAR (BC1cCAR) Construct

We have observed that transduction of compound CAR constructs in generallack high efficiency gene transfer rates compared to single antigenCARs. Whether due to construct size or metabolic stress on effectorcells or other factors, optimization of a transduction schema forcompound CARs remain necessary. We compared 3 different protocols fortransductions and major differences included whether incubation occurswithin viral supernatant, transduction procedure frequency, and finalcell density numbers per treatment. Method 1 was a 2×transduction for 24hours each time and uses retronectin coated plates incubated with virusfirst, aspirated, then incubated with T-cells to a final concentrationof 0.5×10⁶ cells/ml. Method 2 used the same viral retronectin procedure,however, it exchanged the 2^(nd) transduction period for continuedincubation to a total of 48 hours of incubation with a final celldensity of 0.3×10⁶ cells/ml. Method 2 revised uses an incubation schemewhere viral supernatant was directly incubated with cells for 48 hourson a retronectin coated plate at a cell density of 0.3×10⁶ cells/ml(FIG. 23 ).

Transduction Protocol Optimizations Correlate to Improved BC1cCARSurface Expression

BC1cCAR's modular design consists of an anti-CD269 (BCMA) single-chainvariable fragment (scFv) region fused to an anti-CD319 (CS1) scFv by aself-cleaving P2A peptide, CD8-derived hinge (H) and transmembrane (TM)regions, and 4-1BB co-activation domains linked to the CD3ζ signalingdomain (FIG. 24A). A strong spleen focus forming virus promoter (SFFV)and a CD8 leader sequence were used for efficient expression of theBC1cCAR molecule on the T-cell surface. T-cells isolated from humanperipheral blood buffy coats were transduced with BC1cCAR lentivirusafter 2 days of activation. According to the different transductionschemas above, various transduction efficiencies are reported for eachtechnique (FIG. 24B). We find that, in general, cells incubated withviral supernatant for 48 hours at reduced cell densities (0.3×10⁶cells/ml) support the highest gene-transfer efficiencies (FIG. 24B).Thus, as we improve our transduction schemes, we observe acorrespondingly higher rate of gene transfer (FIG. 24C).

Transfected BC1cCAR HEK293T cells were subjected to Western blotanalysis in order to confirm the compound construct. Immunoblot with ananti-CD3ζ monoclonal antibody showed bands of predicted size for thecompound CAR CD3ζ fusion protein (FIG. 24D). Importantly, two distinctbands of similar intensity were observed on the blot signaling thesuccessful high cleavage action of the P2A peptide as expected. No CD3ζexpression was seen for the GFP control vector as expected.

BC1cCAR T-Cells Specifically Lyse BCMA⁺ and CS1⁺ Myeloma Cell Lines

To assess the cytotoxicity ability of BC1cCAR T-cells, we conductedco-culture assays against myeloma cell lines: MM1S (BMCA⁺ CS1⁺),RPMI-8226 (BCMA⁺ CS1⁻), and U266 (BCMA⁺ CS1^(dim)). The ability of theBC1cCAR T-cells to lyse the target cells was quantified by flowcytometry analysis, and target cells were stained with Cytotracker dye(CMTMR). In 24 hour co-cultures, the BC1cCAR exhibited virtuallycomplete lysis of MM1S cells, with over 90% depletion of target cells atan E:T ratio of 2:1 and over 95% depletion at an E:T of 5:1 (FIGS. 25Aand 25C). In RPMI-8226 cells, BC1cCAR lysed over 70% of BCMA⁺ targetcells at an E:T ratio of 2:1, and over 75% at an E:T of 5:1 (FIGS. 25Aand 25C). In 24 hour co-culture with U266 target cells, BC1cCAR lysed80% of BCMA+U266 cells at an E:T ratio of 2:1, reaching saturation(FIGS. 25B and 25C). As the myeloma cell lines are all mostly BCMA+,these results suggest that largely BCMA targeting by BC1cCAR T-cellspromotes effective cell lysis.

BC1cCAR T-Cells Specifically Target BCMA⁺ and CS1⁺ Populations inPrimary Patient Myeloma Samples

We conducted co-cultures using BC1cCAR T cells against primary tumorcells to evaluate their ability to kill diverse primary myeloma celltypes. Flow cytometry analysis of the MM10-G primary sample revealdistinct and consistent BCMA⁺ and CS1⁺ population subsets. MM7-G sampleshows a complete BCMA⁺ CS1⁺ phenotype while MM11-G exhibits a noisyBCMA^(dim)CS1^(dim) phenotype likely attributable to its property ofbeing a bone-marrow aspirate. BC1cCAR T-cells show robust dose-dependentablation of the MM7-G primary patient sample, with over 75% lysis at anE:T ratio of 5:1, increasing to over 85% at 10:1 (FIG. 26A).

BC1cCAR also show targeted and specific lysis ability, by significantlyablating both the BCMA⁺ CS1⁺ and the BCMA-CS1⁺ population subsets inMM10-G co-cultures. At an E:T ratio of 2:1, BC1cCAR T-cells ablate over60% of the BCMA⁺ CS1⁺ population, and 70% of the CS1⁺ only population(FIG. 26B). At an E:T ratio of 5:1, the ablation of CS1⁺ only populationincreases to 80% (FIG. 26B). Against the MM11-G (FIG. 26C), BC1cCART-cells were also able to demonstrate cytotoxic activity in adose-dependent manner as well (FIG. 26C). In summary, BC1cCAR T cellsexhibit robust anti-tumor activity against both myeloma cell lines andprimary tumor cells presenting different combinations of BCMA and CS1(FIG. 26D)

Functional Evaluation of BC1cCAR Antigenic Specific Activity

To assess and characterize the biological properties of the BC1cCAR interms of its antigenic targeting, we established a model that wouldallow us to test the BC1cCAR scFv functionality independently. Using aCML cell line negative for myeloma markers (K562), we established astable CS1 expressing K562 cell line (CS1xpK562) by transducing CS1 cDNAinto K562 cells and subsequently promoting stable expression throughpuromycin selection (FIG. 27A). To test the BCMA scFv functionality, weobtained a BCMA expressing K562 cell line (BCMAxpK562) from the NIH(Kochenderfer Lab). After we confirmed the independent expression ofeach antigen for each antigen expressing cell line (FIG. 27A), we usedthem in co-culture experiments to determine BC1cCAR T targetingfunctionality.

In short-term cultures (<24 hrs), BC1cCAR T-cells exhibited cytotoxicactivity against BCMAxpK562 cells while showing no effect againstwild-type K562 cells (FIG. 27B). Next, short-term cultures againstCS1xpK562 cells show similar responses against CS1 expressing targetcells. Furthermore, BC1cCAR T-cells appeared to have a strongercytotoxic effect than a CS1-specific CAR against CS1xpK562 cells (FIG.27B). Further validation of the anti-CS1 activity was performed onCS1^(dim) expressing NK-92 cells where cytotoxicity exhibited as adose-dependent effect (FIG. 27B).

To model antigen escape in potential clinical scenarios, we conductedcombined co-culture experiments. We mixed BCMAxpK562 and CS1xpK562 in1:1 ratios and looked for evidence of antigen residual populations thatcould lead to relapse in real world scenarios. Co-cultures were carriedout over 48 hours to ensure antigen depletion. Next, histograms wereconstructed that represents populations of T-cells and target tumorcells. The numbers in each histogram plot represents the residual gatedtarget tumor population. We found that compared to control T-cells, aBCMA-specific CAR and a CS1-specific CAR were able to deplete or haveprofound cytotoxic effects against their respective populations.However, a CS1-specific CAR left a significant residual BCMA⁺population, whereas a BCMA-specific CAR achieved a high degree ofcytotoxicity but still left a small but definite CS1⁺ population (FIG.27C). In contrast, the BC1cCAR T-cells effectively depleted both targetpopulations (FIG. 27C). We speculate that residual tumor populationspossessing 1 antigen may lead to relapse in patients that have undergonetreatment using only a single antigen-specific CAR.

Since normal bone marrow expresses a small subset of plasma cells thatcan express CS1, there are concerns that a CS1 directed CAR could beadversely cytotoxic. While the CS1 population in bone marrow is indeedaffected by the BC1cCAR in a dose-dependent manner (FIG. 27D), the CS1subset itself is small.

BC1cCAR T-Cells Exhibit Persistency and Sequential Killing Ability Evenwith Tumor Re-Challenge

We next investigated the ability of BC1cCAR T-cells to kill tumor cellsin a sequential manner under unfavorable microenvironments caused bycell lysis, debris, and tumor re-challenge. Using the scheme in FIG.28A, we conducted long-term co-cultures using MM1S cells as a modelmyeloma tumor and periodically re-challenged BC1cCAR T-cells and otherCAR constructs with fresh MM1S to simulate tumor expansion or relapse.The initial co-culture condition was done at an E:T ratio of 1:1. Withno exogenous cytokines, we find that depletion of target antigens isaccomplished by all CAR cells after 48 hours, with significantclustering and T-cell proliferation (FIG. 28B). In contrast, controlT-cells show no response and proliferation yielding a tumor populationthat has now expanded by twice its initial number. After re-challengingall treatment wells with fresh MM1S cells we find that all CARs stillretain a high degree of cytotoxicity even without exogenous cytokines.By 108 hours, the newly inputted MM1S cells have been virtually depletedby both BCMA-CAR and the BC1cCAR with significant cytotoxicity stillobserved from the CS1-CAR. However, at this stage, flow cytometry show adiminished CS1-CAR population and a relative growth in the tumor antigenpopulation to ˜17% (FIG. 28C), suggesting that the CS1-CAR T-cells maybe faltering. At this time point, the control T-cells have beencompletely overgrown by tumor cells. All CAR tumor-lysis andcytotoxicity stopped after 168 hours, however, BCMA-CAR and BC1cCARstill show detectable minority T-cell populations while control T-cellsand CS1-CAR T-cells have all virtually disappeared (data not shown).

BC1cCAR T-Cells Exhibit Significant Control and Reduction of Tumor InVivo

In order to evaluate the in vivo anti-tumor activity of BC1cCAR T-cells,we developed a xenogeneic mouse model using NSG mice sublethallyirradiated and intravenously injected with luciferase-expressing MM1Scells, a multiple myeloma cell line, to induce measurable tumorformation. Three days following tumor cell injection, mice wereintravenously injected with 5×10⁶ BC1cCAR T-cells or control GFP cellsin a single dose. On days 3, 6, 8 and 11, mice were injectedsubcutaneously with RediJect D-Luciferin (Perkin Elmer) and subjected toIVIS imaging to measure tumor burden (FIG. 29A). Average light intensitymeasured for the BC1cCAR T-cell injected mice was compared to that ofGFP control mice in order to determine the control of tumor growth byBC1cCAR treatment (FIG. 29B). Unpaired T test analysis revealed anextremely significant difference (P<0.01) between the two groups by Day6 with less light intensity and thus less tumor burden in the BC1cCART-cell injected group compared to control (FIG. 29B). Next, we comparedmouse survival across the two groups (FIG. 29C). All of the BC1cCART-cell injected mice survived past day 50 and over a quarter remainedpast day 65. P-value between control and treated mice is 0.0011 based onLong-Rank Mantel-Cox test. The percent survival of control T-cellinjected mice started to decrease shortly by Day 50 and were deceased byDay 55. In summary, these in vivo data indicate that BC1cCAR T-cellssignificantly reduce tumor burden and prolong survival in MM1S-injectedNSG mice when compared to control cells.

BC1cCAR T-Cells Exhibit Improved Cytotoxic Effect in a Mixed AntigenXenogeneic Mouse Model.

To evaluate the dual targeting nature of the compound CAR that maypreclude antigen escape, we designed a xenogeneic mouse model using NSGmice sublethally irradiated and intravenously injected withluciferase-expressing K562 cells expressing either stably transducedBCMA or CS1. BCMA and CS1 expressing K562 cells were further sorted forexpression following puromycin selection and established as stablehomogenous single antigen populations. BCMA and CS1 expressing K562cells were then mixed at a 4:1 ratio respectively before injection tomodel potential antigen escape. Three days following tumor cellinjection, mice were intravenously injected with a course 15×10⁶ controlT-cells, BCMA-specific CAR, or BC1cCAR T-cells. Two control mice died asa result of injection procedure as a result of technical issues duringT-cell infusion and cell aggregation. On days 3, 7, 10 and 12, mice wereinjected subcutaneously with RediJect D-Luciferin (Perkin Elmer) andsubjected to IVIS imaging to visualize tumor burden (FIG. 19D). Averagelight intensity (signifying tumor burden) measured for the BC1cCART-cell injected mice was compared to that of a BCMA-specific CAR and GFPcontrol injected mice in order to determine the control of tumor growthby treatment (FIG. 29D). By day 10, both the BCMA-specific CAR andBC1cCAR T-cells exhibited over 47% tumor reduction compared to control.However, there was a 6% difference in the tumor burden reduction infavor of the BC1cCAR as early as day 10 on the dorsal side of the mice.By day 12, there was a 17% difference in tumor reduction in favor ofBC1cCAR (FIGS. 19D and E) on the dorsal side. This number approaches thepercentage of CS1-K562 cells injected (20%) versus BCMA-K562 (80%). Itis likely the result of CS1 expressing K562 cells surviving andproliferating as a model for antigen escape. In summary, these in vivodata indicate that BC1cCAR T-cells appeared to show improved tumorburden control for multiple antigen populations.

BC1cCAR Transduction and Validation of Anti-Tumor Properties in NK Cells

To further evaluate the robustness of BC1cCAR in different settings, wetransduced the BC1cCAR construct into a model NK cell line, NK-92. Theconstruct was successfully able to be transduced via lentiviralincubation for 48 hours into NK-92 cells and resulted in a surfaceexpression profile of 62.1% after gene-transfer (FIGS. 30A and 30B).Maintenance of NK-92 cells at densities of 0.3-0.5×10⁶ cells/ml resultedin stable populations. To test for BC1cCAR anti-tumor activity in vitro,we conducted co-cultures against myeloma cell lines and a primarypatient sample. The BC1cCAR approached 80% lysis against MM1S, U266, andRPMI-8226 cell lines at E:T ratios of 5:1 in culture. It alsosuccessfully lysed over 60% of the primary MM7-G tumor (FIGS. 31A and31B). These results are similar in terms of comparability with BC1cCART-cells. Next, we assayed the antigen specificity of the BC1cCAR in itsability to lyse BCMA⁺ or CS1⁺ cell independently. Similar assays werecarried out for BC1cCAR T-cells (FIG. 27 ). In 4 hour cultures witheither BCMA expressing K562 (BCMAxpK562) or CS1 expressing K562(CS1xpK562 cells), we find that the BC1cCAR NK cells are able to havecytotoxic effects against either population (FIG. 31B).

Generation of cCAR Including BCMA CD19 or BCMA CD19b for Treatment ofPlasma Cell Myeloma or Autoimmune Disorders

Generation and Characterization of BCMA-CD19 cCAR or BCMA-CD19b cCARConstruct

BC1cCAR's modular design consists of an anti-CD269 (BCMA) single-chainvariable fragment (scFv) region fused to an anti-CD319 (CS1) scFv by aself-cleaving P2A peptide, CD8-derived hinge (H) and transmembrane (TM)regions, and 4-1BB co-activation domains linked to the CD3ζ signalingdomain (FIG. 35 ). A strong spleen focus forming virus promoter (SFFV)and a CD8 leader sequence were used for efficient expression of theBC1cCAR molecule on the T-cell surface and anti-tumor activities invitro and in vivo using a similar approach described above.

Each of units of CAR in the BCMA CD19 CAR will be tested for itsanti-plasma cell or anti-B cell activity. We found that the BCMA CARunit was able to potently lyse any BCMA⁺ population. We first conductedco-cultures against the dual BCMA CS1 positive plasma cell line MM1S andused a CS1 CAR as a secondary measure for robustness. We observed thatboth BCMA and CS1 specific CARs were able to lyse MM1S targets at highefficiency (FIG. 36A). Next, we cultured the BCMA CAR and CS1 CARagainst a majority BCMA⁺ primary myeloma sample MM7-G. We find that,with regard to BCMA expression, the BCMA CAR was able to virtuallydeplete all BCMA+ cells. In contrast, the CS1 CAR left a residual BCMA⁺population (FIG. 36B). These results suggest that a BCMA CAR achieveshigh potency and specificity in its cytotoxic effect.

We next tested the CD19 CAR unit for its anti-B cell activity. Thesingle-chain variable fragment (scFv) nucleotide sequences of theanti-CD19 molecule was used for two different constructs, CD19-2G andCD19b-BB CAR. To improve signal transduction, the CD19CAR was designedwith 4-1BB co-activation domain fused to the CD3zeta signaling domain,making it a second generation CAR (FIG. 37A). CD19-targeting secondgeneration CAR T-cells have previously been used in clinical trials. Forefficient expression of the CD19CAR molecule on the T cell surface, astrong spleen focus-forming virus promoter (SFFV) was used and theleader sequence of CD8a was incorporated in the construct. Forcomparison, CD19CAR constructs using the leader sequences of CD45, CSF,human albumin (HA) or IL-2 were also made. The anti-CD19 scFv wasseparated from the intracellular signaling domains by CD-8 derived hinge(H) and transmembrane (TM) regions (FIG. 37 ). The CD19CAR DNAmolecules, with different leader sequences or different scFv sequences,were also subsequently sub-cloned into a lentiviral plasmid.

Transduced T cells efficiently express CD19CAR-Lentiviral vectorsupernatant was generated from HEK293T cells transfected with CD19-2Gvector construct and control vector. After collection of lentiviralsupernatant was collected, cells were harvested, lysed, andelectrophoresed prior to Western blot transfer. Incubation of blotmembrane with anti-human CD3zeta antibody resulted in a ˜56 kDa band inthe lane containing lysate from cells transfected with CD19-2G, thepredicted size for the expressed fusion protein (FIG. 37B). Next,peripheral blood mononuclear buffy coat cells were activated for threedays and transduced with L8-CD19-2G, or control vector lentiviralsupernatant on non-tissue culture plates coated with retronectin. Thetransduction procedure was repeated 24 hours after the firsttransduction. CAR expression on the T-cell surface was demonstratedthree days after transduction by staining transduced T cells with goatanti-mouse Fab antibody and mouse anti-human CD3. FIG. 37C shows that19.8% of cells transduced with the L8-CD19-2G virus were positive forboth F(Ab′)2 and CD3 as determined by flow cytometry, when compared tothe control transduction.Transduced T cells express CD19-2G at different levels based on leadersequences—To determine the leader sequence that would result in thehighest level of cell surface expression of CD19-2G CAR, severalconstructs were made that incorporated leader sequences for human CD8a(L8), CD45 (L45), colony stimulating factor (CSF), human albumin (HA),and IL2 (FIG. 38A). Following transduction of human peripheral blood Tcells with lentiviral supernatant generated from each of these vectors,transduction efficiency for the T cells was determined using F(Ab′)2antibody as above. Only the CD19-2G construct incorporating the L8leader sequence resulted in any appreciable cell surface expression ofCAR (32.5%), while the L45 leader sequence resulted in only 3.3%transduction efficiency, and CSF, HA and IL2 were below 1% (FIG. 38B).This shows that the optimal design of CD19-2G CAR depends in part on theleader sequence used.Transduced T Cells Express CD19-2G at Different Levels Based on scFvSequencesTo determine the scFv sequence of CD19 that would result in the highestlevel of cell surface expression of CD19-2G CAR, two different sequenceswere used in the design of CD19-2G CAR (FIG. 39A), CD19 and CD19b. Bothused the L8 leader sequence. Following transduction of human peripheralblood T cells with lentiviral supernatant generated from each of thesevectors under the same condition, transduction efficiency for the Tcells was determined using F(Ab′)2 antibody as above. The CD19-2Gconstruct resulted in 18.2% CAR cells, but the CD19b-BB-2G constructresulted in 54.7% CAR efficiency (FIG. 39 b ). This shows that theoptimal design of CD19-2G CAR also depends in part on the sequence ofthe scFv used.

CD19-2G and CD19b-BB-2G CAR T Cells Specifically Target CD19-ExpressingCell Lines

T cell co-culture killing assays were performed to determine the abilityof CD19-2G and CD19b-BB-2G CAR T cells to effectively lyse the CD19+cell lines, SP53 and JeKo-1 (both mantle cell lymphoma lines). Briefly,each target cell line was pre-labeled with CMTMR membrane dye, and thenco-cultured with either vector control, L8-CD19-2G or L8-CD19b-BB-2G CART cells at ratios of 2:1 and 5:1 effector:target cells (200,000 or500,000 effector cells to 100,000 target cells, in 1 mL T cell mediawithout serum or IL-2). After overnight incubation, cells were labeledwith anti-human CD3-PerCp and CD19-APC for 30 minutes, washed, andsuspended in 2% formalin for analysis by flow cytometry. Both CD19-2Gand CD19b 2G CAR T cells displayed robust lysis of B cell lines, SP53and Jeko-1 (FIG. 41 ).

CD19-2G and CD19b-BB-2G CAR T Cells Eliminate CD19+ Cells from AML andB-ALL Patient Samples

Studies were also conducted using patient samples. Two patients withCD19+ cells were used: one diagnosed as AML (aberrant expression ofCD19), and one with B-ALL, were used in the study. The patients' bloodcontained 26.4% and 90% of CD19+ cells, respectively (FIGS. 41A, 42A).Buffy coat fractions of these primary cells were pre-labeled with CMTMRand co-cultured with either vector control, L8-CD19-2G or L8-CD19b-BB-2GT cells in the same manner and ratios as the tumor cell lines. BothL-8-CD19-2G CAR and L-8-CD19b-2G cells were able to complete eliminatethe target cells expressing CD19 (FIGS. 42 and 43 ).

Viral titers generally decrease as the size of insert increases and thesequence of CD19b scFv provided a higher titer for CD19b CAR (FIG. 39 ).Therefore, CD19b scFv was used to generate the compound BCMA CD19b CAR(FIG. 44 ). BCMA CD19b CAR.

An Alternative CAR Design for Myeloma and Plasma Cells

We designed a ligand expressing CAR that binds to various B-cellactivation factor receptors. While it seems a logical leap to designCARs for any potential antigen or ligand factor that can be bound to atumor population, technical troubleshooting in CAR technology is still ahigh and persistent barrier. Not all CAR constructs are able to achieveconsistent or sufficient surface expression as a result of undefinedmolecular interactions or design problems. We were able to achievesurface expression of CD45 leader sequence BAFF-CAR with a CD28intracellular signaling domain of around 21% (FIG. 45A). However,BAFF-CARs with alternate leader sequences from CD8 or CSF did notachieve any meaningful expression (FIG. 45B). Yet another factor wasobserved when CAR design was considered. We designed BAFF-CAR constructsusing the 4-1BBL ligand binding domain as a supportive stimulatorypathway in one case. In another, we added an IL-15/IL-15sushi armorexpressing arm to the construct. The CD8 leader sequence paired with the4-1BBL or the IL-15/IL-15sushi both achieved higher surface expressionthan the CSF leader sequence in both cases (FIG. 45C).

Anti-Plasma Cell Properties of the BAFF-CARs

We characterized the biological properties of the various BAFF-CARs byculturing them with either plasma cell myeloma cells (MM1S) or mantle(MCL) cells (SP53) that all express a component of the plasma cellmarker CD138 to which BAFF is a ligand bound complex. The L45-BAFF-28CAR was able to lyse MM1S tumor cells after 48 hours at an E:T ratio of3:1 approaching 60% (FIG. 46 ). Furthermore, the L8-BAFF-28IL-15/IL-15sushi and L8-BAFF-28 4-1BBL CARs were also able to achievecomparable degrees of cytotoxicity (FIG. 47A, 47C). Co-culture with theB cell mantle cell line SP53 show a limited effect with around 25%cytotoxicity observed for the L8-BAFF-28 IL-15/IL-15 CAR only (FIG. 47).

CD45 CAR Therapy

Three pairs of sgRNA are designed with CHOPCHOP to target the gene ofinterest. Gene-specific sgRNAs are then cloned into the lentiviralvector (Lenti U6-sgRNA-SFFV-Cas9-puro-wpre) expressing a human Cas9 andpuromycin resistance genes linked with an E2A self-cleaving linker. TheU6-sgRNA cassette is in front of the Cas9 element. The expression ofsgRNA and Cas9puro is driven by the U6 promoter and SFFV promoter,respectively (FIG. 48 ).

The following gene-specific sgRNA sequences were used and constructed,

In a non-limiting embodiment of the disclosure, exemplary gene-specificsgRNAs have been designed and constructed as set forth below:

CD45 sgRNA construct:

Lenti-U6-sgCD45a-SFFV-Cas9-puro GTGGTGTGAGTAGGTAALenti-U6-sgCD45b-SFFV-Cas9-puro GAGTTTTGCATTGGCGGLenti-U6-sgCD45c-SFFV-Cas9-puro GAGGGTGGTTGTCAATGFIG. 49A shows steps of generation of CD45 CAR T or NK cell targetinghematologic malignancies.

CRISPR/Cas Nucleases Target to CD45 on NK Cells

Lentiviruses carried gene-specific sgRNAs were used to transduce NK-92cells. The loss of CD45 expression on NK-92 cells was determined by flowcytometry analysis. The CD45 negative population of NK-92 cells wassorted and expanded (FIG. 49B). The sorted and expanded CD45 negativeNK-92 cells were used to generate CD45CAR NK cells. The resultingCD45CAR NK cells were used to test their ability of killing CD45+ cells.

Functional Characterization of CD45 Inactivated NK-92 Cells(NK^(45i)-92) after CRISPR/Cas Nucleases Target

We demonstrated that, following CRISPR/Cas nuclease inactivation ofCD45, the growth of NK^(45i)-92 cells was similar to that of the wildNK-92 cells (FIG. 50 ). Inactivation of CD45 did not significantlyaffect the cell proliferation of NK-92. In addition, we showed that thelysis ability of NK^(45i)-92 cells was compatible to that of wild type,NK-92 when cells were co-cultured with leukemic cells, CCRF (FIG. 51 ).

To demonstrate that CD45-inactivated NK-92 was compatible with CARlysis, NK^(45i)-92 cells and their wild type, NK-92 were transduced withlentiviruses expressing CD5CAR or GFP. The resulting CD5CAR NK^(45i)-92cells and GFP NK^(45i)-92 were sorted by FACS, and used to compare theirability of killing targeted cells. CD5CAR NK^(45i)-92 cells displayedthe ability of robustly killing CD5 target leukemic cells at ratios(E:T), 2:1 and 5:1 when they were co-cultured with CCRF-CEM cells. Weshowed that there was a similar efficacy of elimination of CCRF-CEMcells in vitro between CD5CAR NK^(45i)-92 and CD5 CAR NK-92 cells (FIG.52 ). This suggests that the loss of CD45 expression does not diminishthe anti-tumor activity of CAR NK-cells.

Generation of CD45CAR Construct

We next investigate that CD45CAR in NK^(45i)-92 cells response to theCD45 antigen in leukemic cells. We generated CD45CAR. CD45CAR consistsof an anti-CD45 single-chain variable fragment (scFv) region,CD8-derived hinge (H) and transmembrane (TM) regions, and tandem CD28and 4-1BB co-activation domains linked to the CD3ζ signaling domain(FIG. 53A). A strong spleen focus forming virus promoter (SFFV) and aCD8 leader sequence were used. CD45CAR protein was characterized byWestern blot of HEK293-FT cells transfected with CD45CAR lentiviralplasmid with appropriate vector control. Additionally, anti-CD3zetamonoclonal antibody immunoblots revealed bands of predicted size for theCD45CAR protein with no bands observed in vector control (FIG. 53B).

CD45CAR NK^(45i)-92 NK Cells

Following fluorescence-activated cell sorting (FACS) to enrich forNK^(45i)-92 cells, CD45CAR NK-92 transduction efficiency was determinedto be 87%, as determined by flow cytometry (FIG. 54 ) after sorting.After FACS collection of NK^(45i)-92 cells, CD45CAR expression levelsremained consistently stable for at least 10 passages.

CD45CAR NK^(45i)-92 Cells Specifically Lyse CD45+ Leukemic Cells.

To assess CD45CAR NK^(45i)-92 anti-leukemic activity, we conductedco-culture assays using T-ALL cell lines, CCRF-CEM and Jurkat, and NKcell line and NK-92 cells since they all express CD45 (FIGS. 55, 56 and57 ). We demonstrated that CD45CAR NK^(45i)-92 cells consistentlydisplayed robust lysis of leukemic cells. Following 6-hour incubation ata low effective to target cell (E:T ratio 5:1), CD45CAR NK^(45i)-92cells effectively lysed more than 60% of CCRF-CEMcells (FIG. 55 ). After6-hour co-culture, CD45CAR NK^(45i)-92 cells were also able to eliminateabout 60% of Jurkat cells at a ratio of E:T, 2:1 or 5:1 (FIG. 56 ).After 6 hours of co-culture, CD45CAR NK^(45i)-92 cells efficiently lysed20% CD45 positive NK-92 cells at an E:T ratio of 2:1, with close to 60%lysis at an E:T of 5:1 (FIGS. 57A-57C).

To further analyze the CD45 target for hematologic malignancies, we alsogenerated additional two CARs: CD45-28 and CD45-BB, and the lentivirusesexpressing CD45-28 or CD45-BB CAR were used to transduce NK45i-92 cells.CD45-28 and CD45-BB CARs contain a new anti-CD45 scFv, which isdifferent from that of CD45CAR described above. CD45-28 CAR uses a CD28co-stimulatory domain while the CD45-BB bears a 4-BB co-stimulatorydomain. Both CARs use the CD8-derived hinge (H), transmembrane (TM)regions and CD3ζ signaling domain. CD45CARs displayed robust lysis of Bacute lymphoblastic cell line, REH. CD45CAR NK45i-92 cells lysed about76% REH cells. CD45b-BB CAR NK45i-92 cells and CD45b-28 CAR NK45i-92cells showed about 79% and 100% lysis of REH cells, respectivelycompared to control GFP NK-92 cells (FIG. 57D-57E). CD45b-28 CARNK45i-92 cells exhibited the highest ability of lysis of REH cells(B-ALL cells).

We also investigated if CD4b-28CAR CD45b-28 CAR NK45i-92 cells couldlyse other types of leukemic cells. As shown in FIG. 57F, co-cultureassay was performed with U937 cells (target: T) and GFP NK-92 cells orCD45b-28 NK^(45i)-92 cells (effector: E) at 2:1 (E:T) ratio for 20hours, CD45b-28 NK^(45i)-92 cells exhibited a robust anti-leukemicactivity with about 81% cell lysis against U937 cells compared tocontrol GFP NK-92 cells. U937 is an acute myeloid leukemia cell line. Asimilar finding was seen when co-culture assay was done with MOLM-13cells (target: T) and GFP NK-92 cells or CD45b-28 NK45i-92 cells(effector: E) at 5:1 (E:T) ratio for 20 hours (FIG. 57G). MOLM-13 cellsare derived from a patient with aggressive acute monocytic leukemia. Theanti-leukemic activities were also examined in two mantle cell lines,SP53 and Jeko (FIGS. 57H and I). CD45b-28 NK^(45i)-92 with a low ratioof 2:1 (E:T), were able to lyse more than 40% of SP53 cells or Jekoleukemic cells compared to control GFP NK-92 cells at a relative shortco-culture period of time, 6 hours. These studies demonstrated thatCD45b-28 NK^(45i)-92 had a remarkable anti-leukemic property againstdifferent types of malignant leukemias.

We further investigated if CD45b-28 NK^(45i)-92 cells could lyse CD34+hematopoietic stem/progenitor cells. CD34(+) stem cells derived fromhuman umbilical cord blood were co-cultured with either control orCD45b-28 CAR NK cells for 48 hr at a low ratio of 2:1 (effective:target). CD45b-28 NK^(45i)-92 cells nearly eliminate CD34+ hematopoieticprecursor cells (FIG. 57J) compared to the control.

An Alternative CAR Design to Enhance CD45 CAR Activity

We also generated engineered CD45 CAR cells received not onlycostimulation through the CD28 but also co-express the 4-1BB ligand(4-1BBL or CD137L) in a single construct, which provide the bettertherapeutic efficacy (FIG. 58A) and their example is described below:

Example: CD45b-28-2G-4-1BBL was generated and the generated CD45b CARcells could receive both co-stimulatory pathways, CD28 and 4-1BB.CD45b-28-2G-4-1BBL viruses were concentrated by 4 fold and used totransduce NK^(45i)-92 cells. Its CAR surface expression was about 87%(FIG. 58B). CD45b-28-2G-4-1BBL viruses were concentrated by 4 fold andused for transduction. Anti-tumor activity of CD45b-2G CAR cells wassignificantly improved when 4-1BBL was included in the construct.

An enhancer, IL-15/IL-15sushi was also included in CD45 CAR construct asan alternative approach to enhance CD45 CAR anti-tumor activity. BothCD45 CAR and IL-15/IL-15sushi were in a single construct (FIG. 58 ).Anti-tumor activity of CD45b-2G CAR cells is significantly improved whenIL-15/IL-15sushi is included in the construct.

Example: CD45b-28-2G-IL-15/IL-15sushi NK cells was generated. SurfaceCD45b CAR expression were about 60%. (FIG. 58C). Anti-tumor activity ofCD45b-2G CAR cells was significantly improved when IL-15/IL-15sushi wasincluded in the construct.

Characterization of CD4IL-15/IL-15sushi CAR

The CD4IL-15/IL-15sushi-CAR has been generated and it contains the thirdgeneration of CD4CAR linked to IL-15/IL-15sushi (FIG. 59 ). Acombination of CAR, (third generation), sushi/IL-15 is assembled on anexpression vector and their expression is driven by the SFFV promoter(FIG. 59 ). CAR with IL-15/IL-15sushi is linked with the P2A cleavingsequence. The IL-15/IL-15sushi portion is composed of IL-2 signalpeptide fused to IL-15 linked to IL-15susi via a 26-amino acidpoly-proline linker (FIG. 59 ). The IL-2 signal peptide provide a bettersecreting signal. The stable, functional complexes of IL-15/IL-15sushican be secreted from the transduced cells and the secretion is directedby IL-2 signal peptide.

To verify the CD4IL-15/IL-15sushi construct, HEK293FT cells weretransfected with lentiviral plasmids for either GFP (control) or.CD4IL-15/IL-15sushi. Approximately 60 hours after transfection, bothHEK-293FT cells and supernatant were collected. Cells were lysed in RIPAbuffer containing protease inhibitor cocktail and electrophoresed. Thegel was transferred to Immobilon FL blotting membrane, blocked, andprobed with mouse anti-human CD3z antibody at 1:500. After washes,membrane was probed with goat anti-mouse HRP conjugate, washed, andexposed to film following treatment with HyGlo HRP substrate. TheCD4IL-15/IL-15sushi was successfully expressed in HEK 293 cells (Lane 2,FIG. 60 a ). The CD4IL-15/IL-15sushi lentiviral supernatant was furtherexamined by the transduction of fresh HEK-293 cells (FIG. 60A). HEK-293cells were transduced with either GFP or CD4IL-15/IL-15sushi CAR viralsupernatant from transfected HEK-293FT cells. Polybrene was added to 4μL/mL. Media was changed after 16 hours and replaced with mediacontaining no viral supernatant or polybrene. Three days aftertransduction, cells were harvested and stained with goat-anti-mouseF(Ab′)2 antibody at 1:250 for 30 minutes. Cells were washed and stainedwith streptavidin-PE conjugate at 1:500, washed, suspended in 2%formalin, and analyzed by flow cytometry. FIG. 60 b shows that HEK-293cells that were transduced with the CD4IL-15/IL-15sushi CAR lentiviruswere 80% positive for F(Ab)2-PE (circled, FIG. 60B), while transductionwith GFP control lentivirus was minimal for F(Ab)2-PE (FIG. 60 ).

Production of CD4IL-15/IL-15Sushi-CAR NK Cells

NK-92 cells were transduced with concentrated CD4IL-15/IL-15sushi-CARlentiviral supernatant. After 5 days incubation, cells were harvestedand incubated with goat anti-mouse F(Ab′)2 at 1:250 for 30 minutes.Cells were washed, suspended and stained with streptavidin-PE for 30minutes. Cells were washed and suspended in 2% formalin, and analyzed byflow cytometry, resulting in nearly 70% of the transduced cellsexpressing CD4IL-15/IL-15sushi-CAR (circled, FIG. 61 . Furtherexperimental tests for CD4IL-15/IL-15sushi-CAR includedleukemia/lymphoma killing assays in vitro and vivo, and comparison oftarget killing and proliferation rates with cells transduced withCD4CAR. The inventor also used the same strategy described above togenerate CD19IL-15/IL-15sush CAR, CD20IL-15/IL-15sush CAR andCD22IL-15/IL-15sush CAR.

Production of CD4IL-15/IL-15Sushi-CAR T Cells

Human umbilical cord buffy coat cells were transduced with concentratedCD4IL-15/IL-15sushi-CAR lentiviral supernatant. After 5 days incubation,cells were harvested and incubated with goat anti-mouse F(Ab′)2 at 1:250for 30 minutes. Cells were washed, suspended and stained withstreptavidin-PE for 30 minutes. Cells were washed and suspended in 2%formalin, and analyzed by flow cytometry, resulting in 63% of thetransduced cells expressing CD4IL-15/IL-15sushi-CAR (circled, FIG. 62 ).Further experimental tests for CD4IL-15/IL-15sushi-CAR will includeleukemia/lymphoma killing assays in vitro and vivo, and comparison oftarget killing and proliferation rates with cells transduced withCD4CAR.

CD4IL-15/IL-15Sushi CAR NK Cells were Tested for Anti-Leukemic ActivityRelative to CD4CAR NK Cells In Vitro by Co-Culturing them with theFollowing CD4 Positive Cell Lines: Karpas 299 and MOLT4.

The Karpas 299 cell line was derived from a patient with anaplasticlarge T cell lymphoma. The MOLT4 cell line expressing CD4 wasestablished from the peripheral blood of a 19-year-old patient withacute lymphoblastic leukemia (T-ALL). During 4-hour co-cultureexperiments, CD4IL-15/IL-15sushi CAR NK cells showed profound killing(95%) of Karpas 299 cells at a 5:1 ratio of effector:target, at an evenhigher rate than that of CD4CAR NK cells (82%; FIG. 63 ). Similarly,when co-cultured 1:1 with MOLT4 cells, CD4IL-15/IL-15sushi CAR NK cellslysed target cells at a higher rate (84% to 65%) than CD4CAR NK cells inan overnight assay (FIG. 64 ). These results show that CD4IL-15/sushiCAR NK cells can ablate tumor cells.

Both CD4CAR and CD4IL-15/IL-15Sushi CAR T Cells Exhibit SignificantAnti-Tumor Activity In Vivo

In order to evaluate the in vivo anti-tumor activity of CD4CAR andCD4IL-15/IL-15sushi CAR T cells, and to determine the possible increasein persistence of the CD4IL-15/IL-15sushi CAR T cells relative to theCD4CAR T cells, we developed a xenogeneic mouse model using NSG micesublethally irradiated and intravenously injected withluciferase-expressing MOLM13 cells, an acute myeloid leukemia cell linethat is 100% CD4+, to induce measurable tumor formation (FIG. 65 ).Three days following tumor cell injection, 6 mice each wereintravenously injected with a course of 8×10⁶ CD4CAR,CD4IL-15/IL-15sushi T cells or vector control T cells. On days 3, 6, 9and 11, mice were injected subcutaneously with RediJect D-Luciferin(Perkin Elmer) and subjected to IVIS imaging to measure tumor burden(FIG. 65B). Average light intensity measured for the CD4CAR andCD4IL-15/IL-15sushi CAR T cell injected mice was compared to that ofvector control T cell injected mice in order to determine the percentageof tumor cells in treated versus control mice (FIG. 65C). CD4CAR Tcell-treated mice had a 52% lower tumor burden relative to control onDay 6, whereas CD4IL-15/IL-15sushi CAR T cell-treated mice had a 74%lower tumor burden. On Day 11, nearly all tumor cells had been lysed inboth of these groups. Unpaired T test analysis revealed a verysignificant difference (P=0.0045) between control and the two groups byday 9 with less light intensity and thus less tumor burden in the CD4CARand CD4IL-15/IL-15sushi CAR T cells treated group compared to control.In summary, these in vivo data indicate that CD4CAR andCD4IL-15/IL-15sushi CAR T cells both significantly reduce tumor burdenand in MOLM13-injected NSG mice when compared to vector control T cells.

Next, we compared mouse survival across the two groups (FIG. 65D). Allleukemic mice injected with CD4IL-15/IL-15sushi CAR T cell survivedlonger than that of CD4CAR T 20 cells. In summary, these in vivo dataindicate that CD4IL-15/IL-15sushi CAR T cells significantly reduce tumorburden and prolong survival in CD4IL-15/IL-15sush CAR T-injected NSGmice when compared to control cells.

CD4IL-15/IL-15Sushi CAR NK Cells Exhibit Robust and PersistentAnti-Tumor Activity In Vivo

In order to further evaluate the CD4IL-15/IL-15sushi CAR function, wecreated a stressful condition utilizing NK CAR cells and Jurkat tumorcells. The NK cells bear a short half-life property and leukemic Jurkatcells show less than 60% CD4+ phenotype (FIG. 66A). In such a condition,it allows us to investigate how secretory soluble IL-15sushi affects theCAR functions in terms of its persistence and killing capability. Wethen used our xenogeneic NSG mouse model using NSG mice sublethallyirradiated and intravenously injected with luciferase-expressing Jurkatcells to induce measurable tumor formation. In contrast with MOLM-13cells, Jurkat cells show less than 60% CD4+ phenotype (FIG. 66A5). Threedays following Jurkat cell injection, mice were intravenously injectedwith a course of 10×10⁶ either CD4CAR, CD4IL-15/IL-15sushi, or vectorcontrol NK cells. On day 3 (the day before treatment), 7, 10, and 14,mice were subjected to IVIS imaging to measure tumor burden (FIG. 66B).Average light intensity measured for the CD4CAR and CD4IL-15/IL-15sushiNK injected mice was compared to that of vector control NK injected miceto determine percent lysis of Jurkat cells (FIG. 66C). Although bothconditions showed significant tumor cell lysis by Day 7, lysispercentage for CD4CAR NK cells stayed the same to Day 14 whileCD4IL-15/IL-15sushi NK cells increased to over 97%. (FIG. 66D). UnpairedT test analysis revealed an extremely significant difference (P<0.0001)between the two groups by Day 14. These results indicate that CD4CAR NKcell lysis of Jurkat tumor cells was not able to keep up with theexpansion of CD4− Jurkat cells, whereas the continued expansion of NKCAR cells secreting IL-15/IL-15sushi effectively lysed. Theco-expression of secretory IL-15/IL-15sushi with CAR could supplementthe defect that CAR T or NK cells are unable to eliminate dim expressedcancer cells or non-targeting cancer cells. A repeat of experiments(FIG. 67 ) showed similar results to those described in FIG. 66 .

Secreted IL-15/IL-15Sushi Substitutes for IL-2 in NK Cell Survival andExpansion.

The effect of IL-15/IL-15sushi-secreting NK cells on cell survival wasdetermined. NK-92 cells stably transduced with either CD4CAR orCD4IL-15/IL-15sushi were cultured in the presence or absence of IL-2 todetermine if IL-15/IL-15sushi secretion alone could lead to survival andexpansion. CD4CAR-expressing NK cells cultured without IL-2 died by Day7, while CD4IL-15/IL-15sushi-expressing NK cells cultured without IL-2expanded at approximately the same rate as either CD4CAR orCD4IL-15/IL-15sushi cells cultured with IL-2 (FIG. 68B), showing thatsecreted IL-15/IL-15sushi could substitute for IL-2. Furthermore, wewere able to demonstrate that NK cells secreting IL-15/IL-15sushi couldaid in the survival and expansion of non-transduced NK-92 cells in aco-culture. In this experiment, an equal ratio of NK GFP-expressingcells were cultured with either CD4CAR- orCD4IL-15/IL-15sushi-expressing NK cells, in the presence or absence ofIL-2. Cells were counted every 2-3 days (FIG. 8A). By Day 7, CD4CAR NKcells given no IL-2 had died, but CD4IL-15/IL-15sushi NK cells withoutIL-2 had survived and expanded at approximately the same rate as eitherCD4CAR or CD4IL-15/IL-15sushi cells cultured with IL-2. The number ofGFP-expressing cells had risen along with the CD4IL-15/IL-15sushi NKcells (FIG. 68B), indicating that the secreted IL-15/IL-15sushi hadpositively affected GFP NK cell survival. The percentage of GFP-positivecells had risen from 50% to over 70% over the course of the experiment(data not shown). In the second experiment (FIG. 69 ), GFP NK and eithercontrol vector NK or CD4IL-15/IL-15sushi NK cells were mixed at a 10 to1 ratio, with no IL-2. By Day 6, cells co-cultured with control cellshad all died, but survival of cells cultured with NK cells secretingIL-15/IL-15sushi survived until Day 10.

To further determine if this effect was due to secreted protein alone,or an interaction between co-cultured cells, we devised an experiment inwhich the GFP NK cells were cultured in a chamber above the culturedCD4CAR or CD4IL-15/IL-15sushi NK cells, or non-transduced NK-92 cells.In this situation, only proteins and not cells could pass between themembrane separating the two cultures. Cells were incubated without IL-2,counted and split 1:1 every other day. While GFP NK cells in the upperchamber above NK-92 cells had died by Day 6, the GFP NK cells above theCD4IL-15/IL-15sushi NK cells had survived and expanded by Day 12 (FIG.70 ), thereby indicating that it was the IL-15/IL-15sushi proteinsecreted by the CD4IL-15/IL-15sushi NK cells which had kept them alive,and not direct cell-to-cell contact. In this model, the upper chamberrepresents the tumor microenvironment, in which the survival of T cellsor NK cells is improved by the secretion of IL-15/IL-15sushi from theCD4IL-15/IL-15sushi NK cells.

Effect of Secreted IL-15/IL-15Sushi on CAR T and Non-TransducedNeighboring Cells.

We also compared the cell growth of CD4CAR and CD4IL-15/IL-15sushitransduced T cells in the presence or absence of IL-2. Total cell countscalculated throughout the experiment (up to Day 17) for transduced cellswith or without IL-2. CD4IL-15/IL-15sushi transduced T cells appeared tobe more tolerant to the absence of IL-2 than that of CD4CAR transduced Tcells.

EXAMPLES Generation of Treg CAR Target Treg Cells

Treg CAR (also called CD4zetaCD25CAR or C4-25z) followed the schematicin FIG. 70 . It comprises of SFFV (spleen focus-forming virus) promoterthat drives the expression of two different units of incomplete CARslinked by a P2A cleavage peptide. The CD4 chimeric antigen receptorpolypeptide unit comprises a CD45 signal peptide, a CD4 antigenrecognition domain, a hinge region (derived CD8a), a transmembranedomain (CD8a) and CD3 zeta chain; CD25 chimeric antigen receptorpolypeptide unit comprises a CD45 signal peptide, a CD25 antigenrecognition domain, a hinge region (CD8a), a transmembrane domain(CD8a), a co-stimulatory domain (s), CD28. The Treg CAR can potentiatethe lysis activity of a cell co-expressing CD4 and CD25 while minimizinga cell bearing CD4 or CD25 antigen alone.

The CD4zetaCD25CAR (C4-25z) (Treg CAR) was transduced in an assay.Compared to control T-cells, CD4zetaCD25CAR cells show ˜15% surfaceexpression and this was sufficient to observe the following phenotypevalidation of construct function (FIG. 70A). CD4zetaCD25 CAR cells andcontrol T-cells were both assayed with CD4 and CD25 antibody to look forlogic gated behavior using flow cytometry analysis. Due to the constructdesign, the CD4zetaCD25CAR cell would potentiate the lysis activity forcells co-expressing both CD4 and CD25 antigens. Here, we showeddepletion (˜95%) of the CD4+ CD25+ double positive population withlittle impact of off-logic events in the other phenotype cases. A bargraph summary shows that the logic gated CAR construct design onlysignificantly impacts the double positive population (FIG. 70B).

We further characterized CD4zetaCD25 CAR by comparing it with CD4 CAR.As expected, CD4CAR T cells had a profound lysis ability of cellsexpressing CD4 only while CD4zetaCD25CAR T cells had a limited killingability on this population (FIG. 71 ). CD4zetaCD25CAR T cells alsoshowed virtually complete depletion of cells expressing both CD4 andCD25 antigens (FIG. 71 ). These studies demonstrate that the robustCD4zetaCD25CAR targeting cells co-expressing both CD4 and CD25, has beenestablished. Due to human-specific CD4 or CD25scFv in the construct, thefunctional properties of CD4zetaCD25CAR are difficult to test inanimals.

In some embodiments, the disclosed disclosure also comprises methods ofimproving the CD4zetaCD25CAR therapeutic activity. The example isdescribed below.

Example

An engineered CD4zetaCD25CAR cell was prepared in accordance with thepresent disclosure.

Cell killing assay is performed

Targeted Cells killing by CD4zetaCD25CAR is improved when co-expressedwith 4-1BBL or IL-15/IL-15sushi or IL-15/IL-15RA.

Safety Switch

Introduction of a “safety switch” greatly increases safety profile andthe “safety switch” may be an inducible suicide gene, such as, withoutlimiting, caspase 9 gene, thymidine kinase, cytosine deaminase (CD) orcytochrome P450. Other safety switches for elimination of unwantedmodified T cells involve co-expression of CD20 or CD52 or CD19 ortruncated epidermal growth factor receptor in T cells.

Example: Co-Expression of CD52 with CARs Using CD5CAR Targeting T-CellMalignancies as an Example

For clinical treatment using CAR T-cells against T-cell malignancies,establishment of safety methods to eliminate CAR T-cells from patientsmay be necessary after tumor depletion or in emergency cases due tounexpected side effects caused by CAR therapy. T-cells and B-cellsexpress CD52 on the cell surface and a CD52 specific antibody, CAMPATH(alemtuzumab), can eliminate CD52+ cells from circulation. We thusincorporated a human CD52 sequence into the CD5CAR vector construct(FIG. 72A). This additional CD52 construct mechanically separates thesignaling from native CD52. The aim was to preempt the possibility ofnative CD52 antigen escape on CAR T-cell surface after CAMPATHtreatment. CD5CAR-CD52 lentiviral protein and expression were confirmedvia western blot and flow cytometry analysis using CD52 antibody ontransduced HEK293 cells. We also found that co-expressing CD52 wouldaffect the CAR T cell functions.

In Vivo Depletion of Infused CD5CAR-CD52 T Cells Following Treatmentwith CAMPATH

To assess the effect of CAR elimination by CAMPATH (alemtuzumab)treatment, we conducted in vivo procedures as described (FIGS. 72B and72C). We intravenously injected 5×10⁶ CD5CAR-52 T-cells into irradiatedmice. Next day, we added 0.1 mg/kg of CAMPATH or PBS via IP injectionfor 3 mice of each group. After 6 and 24 hours following CAMPATHtreatment, we collected peripheral blood from the mouse tail anddetermined presence of CD5CAR-52 T-cells by FACS analysis. CAMPATHinjection virtually completely deplete CD5CAR-CD52 T-cells in blood atboth 6 h and 24 h (FIG. 72C). Five days following CAMPATHadministration, CD5CAR-CD52 cells were also completely depleted in boththe bone marrow and spleen (FIG. 72D). These findings support the use ofCAMPATH as a useful strategy in acting as a safety trigger to depleteCAR-T cells from circulation and lymphoid organs.

In one embodiment, the engineered cell includes a CD5 chimeric antigenreceptor polypeptide and an anchor CD52 (SEQ ID NO.70), andcorresponding polynucleotide (SEQ ID NO. 69). In some embodiments, CD52is incorporated into CD5 CAR engineered cell or any CAR engineered celland can be used as a “safety switch” for CAR therapy.

Promoter Testing Using the GFP Reporter

HEK293FT cells were transfected with lentiviral plasmids expressing GFPunder the SFFV, EF1 or CAG promoters. Approximately 60 hours aftertransfection, supernatant was collected from each. Relative viral titerwas determined by first transducing HEK293 cells with supernatant fromeach of the 3 promoters. HEK-293 cells were transduced with GFP viralsupernatant from each of the 3 transfected HEK-293FT cells. Polybrenewas added to 4 μL/mL. Media was changed after 16 hours and replaced withmedia containing no viral supernatant or polybrene. Three days aftertransduction, cells were harvested and washed, suspended in 2% formalin,and analyzed by flow cytometry for GFP expression (FITC). GFP expressionwas seen in each sample, but was highest for the cells transduced withvirus made using the SFFV promoter.

Activated human umbilical cord buffy coat cells were transduced with GFPlentiviral supernatant (amount based on the results of the HEK293transduction efficiency) from each of the promoters. After 5 daysincubation, cells were harvested, washed and suspended in 2% formalin,and analyzed by flow cytometry for GFP expression. 43% of cellsexpressed GFP at high levels (>103) while GFP-expression for cellstransduced with virus using promoters EF1 (15%) and CAG (3%) wereconsiderably lower. Five days later, cells analyzed the same way showednearly the same percentages for each (46%, 15% and 3%, respectively).These results indicate that SFFV promoter leads to stronger expressionthan EF1 or CAG promoters, and that the expression remains high for atleast 10 days post-transduction. Further experimental tests will includelonger incubation times for transduced cells beyond the 10-day window.

Functional Titer of Viral Vector Particles in Supernatants (the % GFPCells as Determined by Flow Cytometry Allows for Proxy Viral TiterAdjustments as Higher Titer Virus Infiltrates More Cells, Leading toHigher % GFP Cell Populations).

To determine functional titer of viral vector particles in each of oursupernatants, HEK 293 cells were transduced with either EF1-GFP orSFFV-GFP viral supernatant, with either 30 μL (low), 125 μL (medium), or500 μL (high) per well of a 12 well tissue-culture treated plate.Culture media was changed the following morning to DMEM plus 10% FBS(FIG. 73 ).

Transduced cells were then trypsinized, washed, and suspended informalin and subjected to flow analysis. The percentage of GFP+ cells ineach of the conditions was determined by flow cytometry using the FITCchannel (FIG. 74 ). In each case, the percentage of GFP+ was higher incells transduced with SFFV-GFP than the cells transduced with thecorresponding volume of EF1-GFP viral supernatant (50% to 18% for low,80% to 40% for medium, and 82% to 70% for high). From this, wedetermined that using the highest volume of EF-promoter virus wascomparable to using the lowest volume of SFFV-promoter virus in terms oftiter, and would allow for comparison of relative promoter strengths forthe following transduction experiments

Transduced cells were also visualized on an EVOS fluorescent microscopeusing GFP at 20× at the same exposure conditions for each well (FIG. 73). Cells transduced with SFFV-GFP viral supernatant were dramaticallybrighter than cells transduced with EF1-GFP. Furthermore, 25 comparingthe image of the EF1-promoter under high viral volume loads with theimage of the SFFV-promoter using low viral volume loads show similarfluorescent intensity. This suggests that the SFFV promoter is astronger driver of gene expression.

Comparison of surface expression and persistence of different promotersin primary T-cells (The % GFP cells as determined by flow cytometry forT-cell transductions show expected differences in GFP cell populationsas expected from the prior experiments on HEK293 cells)

To determine promoter transduction efficiency and persistence of surfaceexpression in primary T cells, activated cord blood buffy coat T cellswere transduced with either 50 μL of SFFV-GFP or 1 mL of EF1-GFP EF1-GFPviral supernatant, in 12-well tissue culture-treated plates pre-coatedwith retronectin (Clontech). Following two overnight transductions,cells were cultured on T cell media with 300 IU/mL IL-2 (Peprotech) andmaintained at 1.0-4.0×10⁶/mL. Cells were washed, suspended in formalin,and subjected to flow cytometry analysis, using the FITC channel todetermine the percentage of GFP+ cells, on 7, 14, 21 and 28 days aftertransduction. The percentage of GFP+ cells was consistently higher for Tcells transduced with SFFV-GFP compared to EF1-GFP-transduced T cells(FIG. 75A), even as the percentage of total GFP+ cells decreased overthis period. A further comparison showed that T cells transduced withthe higher (1 mL) amount of EF1-GFP supernatant actually decreased inpercentage relative to the percent of GFP+ cells transduced with thelower amount (50 μL, or 20-fold less) of SFFV-GFP, between Day 7 and Day28, from over 60% to under 40% (FIG. 75B). This suggests thattransduction using the SFFV promoter led to greater persistence oftransduced cells.

Methods of generating the CAR gene including at least one of a T antigenrecognition moiety (at least one of CD4, CD8, CD3, CD5, CD7, and CD2, ora part or a combination thereof), a hinge region and T-cell activationdomains is provided.

Methods of generating multiple units of CARs (cCAR) targeting antigen(s) including at least one of CD33, CD123, CD19, CD20, CD22, CD269, CS1,CD38, CD52, ROR1, PSMA, BAFF, TACI, CD138, and GPC3, or a part or acombination of a hinge region and T-cell activation domains is provided.

The provided methods also include: 1) generating of the CAR T or NKcells targeting leukemias and lymphomas expressing CD45 and avoidingself-killing; 2) generation of “armored” CAR T or NK cells designed toboth overcome the inhibitory tumor microenvironment and exhibit enhancedanti-tumor activity and long-term persistence.

The present disclosure is not limited to the embodiments described andexemplified above, but is capable of variation and modification withinthe scope of the disclosure and claims.

While there have been described what are presently believed to be thepreferred embodiments of the present disclosure, those skilled in theart will realize that other and further changes and modifications may bemade thereto without departing from the spirit of the disclosure, and itis intended to claim all such modifications and changes as come withinthe true scope of the disclosure.

Various terms relating to aspects of the disclosure are used throughoutthe specification and claims. Such terms are to be given their ordinarymeaning in the art, unless otherwise indicated. Other specificallydefined terms are to be construed in a manner consistent with thedefinition provided herein.

INCORPORATION OF SEQUENCE LISTING

A Sequence Listing conforming to the rules of WIPO Standard ST.26 ishereby incorporated by reference. Said Sequence Listing has been filedas an electronic document via PatentCenter encoded as XML in UTF-8 text.The electronic document, created on Jun. 6, 2023, is entitled “2541-3PCTII/US/CON_ST26.xml”, and is 319,488 bytes in size.

1-104. (canceled)
 105. An engineered T cell or NK cell comprising anucleotide sequence comprising from 5′ to 3′ a first polynucleotideencoding a first chimeric antigen receptor polypeptide (CAR), anucleotide encoding a first self-cleavage peptide, and a secondpolynucleotide encoding a second chimeric antigen receptor polypeptide(CAR), wherein: (i) the first CAR comprises a first signal peptide, afirst antibody binding domain, a first hinge region, a firsttransmembrane domain, a first co-stimulatory domain, and a firstsignaling domain; and (ii) the second CAR comprises a second signalpeptide, a second antibody binding domain, a second hinge region, asecond transmembrane domain, a second co-stimulatory domain, and asecond signaling domain; wherein the first antibody binding domain andthe second antibody binding domain are different from each other andeach bind to a different target, wherein the targets of the first andsecond antibody binding domains irrespective of order are CD7 and CD19,CD7 and BCMA, and CD7 and CD5, wherein the first and secondco-stimulatory domains are intracellular, and wherein the cleavage siteis selected from the group consisting of porcine teschovirus-1 2A (P2A),thoseaasigna virus 2A (T2A), equine rhinitis A virus (ERAV) 2A (E2A),and FMDV 2A (F2A).
 106. The engineered T cell or NK cell according toclaim 105, wherein the T-cell is a CD4 T-cell or CD8 T-cell.
 107. Theengineered T cell or KK cell according to claim 105, wherein the NK cellis a NKT cell or NK-92cell.
 108. The engineered T cell or NK cellaccording to claim 105, further comprising a nucleotide encoding anenhancer, wherein the enhancer is selected from the group consisting ofPD-1, PD-L1, CSF1R, CTAL-4, TIM-3, TGFR beta, IL-2, IL-6, IL-7, IL-12,IL-15, IL-17, IL-18 IL-21, functional fragments thereof, andcombinations thereof, and wherein the enhancer is attached to either endof the 5′ to 3′ nucleotide sequence via a nucleotide encoding a secondself-cleavage peptide.
 109. The engineered T cell or NK cell accordingto claim 108, wherein said enhancer is IL-15 or IL-15/IL-15sushi.
 110. Amethod of reducing the number of cells having at least one cell surfaceantigen selected from the group consisting of CD7, CD19, BCMA (CD269)and CD5 in a patient in need thereof, said method comprisingadministering to said patient an engineered T cell or NK cell accordingto claim 105.